Methods for improving the design, bioavailability, and efficacy of directed sequence polymer compositions via serum protein-based detection of directed sequence polymer compositions

ABSTRACT

There exist in the art methods of detecting simple peptides. However, methods to determine the effective plasma concentration of directed sequence polymers (DSPs), are complicated because DSPs are complex mixtures of peptides, as opposed to individual peptides with a defined amino acid sequence. This application provides improved methods of detecting and assessing DSP compositions, methods for the detection and quantitation of DSP compositions, means to determine and enrich a subset of peptides in a DSP composition based on the subset&#39;s interactions with certain capture polypeptides, and methods for administering DSP compositions to a subject in need thereof, wherein the dosage regimen and quantity may be determined or evaluated based on the above-mentioned methods for detection and quantitation.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/281,470, filed Nov. 17, 2009, and U.S. Provisional Application No.61/386,909, filed Sep. 27, 2010.

BACKGROUND

Complex peptide mixtures are an emerging class of peptide therapeutics,of which Copaxone (glatiramer acetate) is a leading example. Complexpeptide mixtures include diverse peptides with one or more sharedcharacteristics (such as amino acid composition and/or sequencesimilarity) and include altered peptide ligands (APLs), peptide pools,peptide libraries, random sequence polymers (RSP) compositions (e.g.,glatiramer acetate, and compositions disclosed in WO 03/029276, WO05/112972, and WO 05/085323), and directed sequence polypeptide (DSP)compositions (see, for example, WO 2007/120834, WO 2009/051797, and WO2009/128948). DSP and RSP compositions are alike in that both comprise alarge number of different peptides whose sequences vary randomly withincertain defined common parameters. RSP compositions are mixtures ofamino acid polymers (typically linked via peptide bonds) comprising twoor more randomly ordered amino acid residues in various ratios. In RSPcompositions, the sequence similarity springs from the restricted aminoacid content of the peptides, because all peptides consist of the samefew amino acids, arranged in a random order. In DSP compositions, thesequence similarity springs from a shared base peptide sequence, withcertain amino acid positions substituted randomly by a restricted slateof amino acids at defined frequencies.

There exist in the art methods of detecting simple peptides, but fewermethods are suitable for detecting and measuring complex peptidemixtures. Methods to determine the effective plasma concentration ofdirected sequence polymers (DSPs), are complicated because DSPs arecomplex mixtures of peptides, as opposed to individual peptides with adefined amino acid sequence. Improved methods for evaluating theconsistency and composition of DSPs through multiple manufacturingpreparations are needed. Determining the in vivo status of DSPcompositions has immunologic significance because, depending on theroute and/or frequency of administration and the serum proteins thatbind the DSPs, a mixture can invoke primarily inflammatory (TH1 type) orprimarily regulatory (TH2 type) responses, leading to variations inpharmacokinetic and pharmacodynamic effects in the subject. Morerigorous design and consistent administration of a DSP composition mayincrease the therapeutic efficacy, or reduce the potential for adverseinflammatory responses.

Thus, there is a need for methods of quantitative analysis of DSPcompositions, e.g., to facilitate the in vivo evaluation of suchmixtures and to determine the suitable amount and means ofadministration for therapeutic purposes.

SUMMARY OF THE INVENTION

This application provides improved methods of detecting and assessingDSP compositions. The instant invention provides methods for thedetection and quantitation of DSP compositions. The instant inventionprovides a means to determine and enrich a subset of peptides in a DSPcomposition based on the subset's interactions with certain capturepolypeptides. The instant invention further provides methods foradministering DSP compositions to a subject in need thereof, wherein thedosage regimen and quantity may be determined or evaluated based on theabove-mentioned methods for detection and quantitation.

The present disclosure also provides a method for detecting a DSPcomposition comprising the steps: (a) affixing said DSP composition to asolid support; (b) contacting said solid support in (a) with aprotein-containing biological fluid; (c) identifying the proteins from(b) specifically bound to the solid support in (a); (d) obtainingsubstantially pure preparations of bound proteins in (c); (e) affixingsaid proteins in (c) to a means for quantitatively detecting said DSPcomposition; and (f) determining binding of said DSP composition to eachindividual said protein in (e).

This disclosure also provides a method for improving the design of a DSPcomposition comprising the steps: (a) affixing said DSP composition to asolid support; (b) contacting said solid support in (a) with aprotein-containing biological fluid; (c) identifying the proteins from(b) specifically bound to the solid support in (a); (d) obtainingsubstantially pure preparations of bound proteins in (c); (e) affixingsaid proteins in (c) to a means for quantitatively detecting said DSPcomposition; (f) determining binding of said DSP composition to eachindividual said protein in (e); (g) adjusting the design of said DSPcomposition to either enhance or reduce binding to one or more proteinsin (e); (h) repeating step (f); (i) optionally repeating steps (f-h),wherein the adjustments to design of said DSP composition results in anyone or more of the group comprising: increased bioavailability,reduction in toxicity, and increase in efficacy.

Furthermore, this application provides a method for detecting specieswithin a DSP composition comprising the steps: (a) affixing said DSPcomposition to a solid support; (b) contacting said solid support in (a)with a protein-containing biological fluid; (c) identifying the proteinsfrom (b) specifically bound to the solid support in (a); (d) obtainingsubstantially pure preparations of bound proteins in (c); (e) affixingsaid proteins in (c) to a solid support; (f) contacting said solidsupport in (e) with said DSP composition; and (g) determining binding ofindividual species of said DSP composition to said solid support in (f).

In addition, this application provides a method for improving the designof species within a DSP composition comprising the steps: (a) affixingsaid DSP composition to a solid support; (b) contacting said solidsupport in (a) with a protein-containing biological fluid; (c)identifying the proteins from (b) specifically bound to the solidsupport in (a); (d) obtaining substantially pure preparations of boundproteins in (c); (e) affixing said proteins in (c) to a solid support;(f) contacting said solid support in (e) with said DSP composition; (g)determining binding of individual species of said DSP composition tosaid solid support in (f); (h) adjusting the design of said DSPcomposition to either enhance or reduce binding to one or more proteinsin (f); (i) repeating step (g); and (j) optionally repeating steps(g-i), wherein the adjustments to design of a species of said DSPcomposition result in any one or more of the group comprising: increasedbioavailability, reduction in toxicity, and increase in efficacy.

Using the methods of the instant application, investigators can not onlymore reliably detect lower amounts of components of the DSPcompositions, but also specifically detect species within DSPcompositions that are responsible for or contribute towards a biologicalactivity of interest, for example toxicity or efficacy.

A finding that underlies the instant invention is the specific bindingof a single peptide or a multiplicity of peptides within a YEAK or YFAKpeptide composition by certain proteinaceous materials. Theproteinaceous materials, herein termed “capture polypeptides”,preferably also bind DSP compositions. Conversely, once the “capturepolypeptides” are identified, one or more of the capture polypeptidescan be used to quantitatively analyze peptides of a DSP composition,isolate functionally superior subsets of the peptides within the DSPcomposition, or classify components of the DSP composition based on thebinding specificity. To practice the instant invention, a capturepolypeptide that binds to the peptides is identified and prepared in aform useful to practice the instant invention, i.e., isolated andpurified to a sufficient degree that its binding to the peptides is notcompromised by the presence of other components.

An aspect of the instant invention provides a method to assess ordetermine variations in the products of distinct manufacturingpreparations, different methods of manufacture, or differentpost-manufacturing processing methods of a DSP composition. A particularmethod of the invention is to compare the binding of differentpreparations of a DSP composition to a capture polypeptide to determinethe similarities and/or differences between preparations.

A further aspect of the invention provides a method to quantitativelyanalyze peptides that are found in a DSP composition or a samplecomprising a DSP composition. Some embodiments of the invention aremethods to determine a biologically available quantity or concentrationin vivo (e.g., a plasma concentration) of an administered DSPcomposition.

A method of the instant invention is to detect the presence of DSPcompositions in a subject's tissue, said subject having previously beenin contact with or treated with the DSP composition, wherein the methodis carried out one or more times immediately after such contact, orafter at least about 10, 20, 30, or 45 minutes, or 1, 2, 4, 6, 12, 24,36, or 48 hours, or 3, 4, 5, 6, 7, or 10 days, or 2, 3, 4, 6, 8, or 12weeks after such contact. A particular method of the instant inventionis to detect the presence of constituents of a DSP composition in theserum or plasma of a mammal, said mammal having been previously treatedwith said DSP composition prior to carrying out said method within atime period described above. In certain embodiments, said mammal is ahuman.

In certain embodiments, the method comprises determining the presenceof, and optionally the quantity of, DSP compositions by binding the DSPsto one or more predetermined capture polypeptides followed by adetection method, such as an immunologic detection method. Thus, oneaspect of the invention comprises selecting or identifying a serumprotein that preferentially binds a DSP composition. In certainembodiments, a method of identifying one or more serum protein comprisescontacting a DSP composition with a biological sample comprising serum,detecting the binding, if any, of peptides in the DSP composition to oneor more components of the serum, isolating the bound components, andidentifying one or more of the bound components. In some embodiments,the bound components can be isolated by contacting the sample with anaffinity column designed to bind the peptides of the DSP composition andsubsequently eluting the bound fraction, followed by identifying thebound component(s).

Any serum binding proteins that bind peptides of DSP compositions can beused in the above methods. Suitable detection methods include DirectCompetitive Enzyme-Linked Immunosorbent Assay (ELISA), Western blot,immunoflow cytometric detection, radioimmunoassay (RIA), or any otherimmunologic detection method that allows quantitative detection ofspecific antigens.

One aspect of the instant invention is a method for detecting thepresence of a DSP composition in a biological sample, comprising:contacting the biological sample with at least one capture polypeptide;and detecting the presence or absence of binding of the capturepolypeptide to the DSP composition, wherein the presence of bindingindicates the presence of peptide components of the DSP composition inthe biological sample. Further, such method can be extended to measurethe amount or concentration of a DSP composition in a sample.

Another aspect of the instant invention is a method for measuringbioavailability of a DSP composition in a mammal, comprising:administering to a mammal a dose of a DSP composition; removing abiological sample from the subject; and contacting the biological samplewith at least one capture polypeptide; thereby determining thebioavailability, or the degree of bioavailability, of the DSPcomposition in the biological sample.

Another aspect of the instant invention provides methods ofadministering DSP compositions to a mammalian subject, such amountdetermined based on the bioavailable portion of the dosed amount asdetermined by the method described above or other methods describedherein. In certain embodiments, the method further comprises including acontrol sample, performing a pharmacodynamic test to determine changesof physiological markers, such as hormones, enzymes, serum proteins,cytokines, immunomodulators, or an effector or regulator of any of thesefunctional proteins, between the control sample and test samples bycomparing the two results, and determining the dosage effective toinduce the desired changes in a pharmacodynamic parameter. In certainembodiments, behavioral changes, subjective changes as reported by asubject such as amelioration of pain or a symptom of a disease, or otherevidence of indirect effects are observed. In certain embodiments, saidmammalian subject is a rodent, such as a mouse or rat. In otherembodiments, said subject is human.

Certain embodiments of this aspect of the invention provide a method fordetermining a suitable dose of a DSP composition to administer to asubject in need thereof, comprising: (a) administering to the subject adose of the DSP composition; (b) removing a biological sample from thesubject; (c) contacting the biological sample with at least one capturepolypeptide; (d) determining a level of components of the DSPcomposition in the biological sample; (e) optionally repeating steps (a)through (d) using a different dose; and (g) comparing the levels to apredetermined suitable level of the DSP composition in the biologicalsample; wherein the suitable dose is a dose that results in thepredetermined suitable level of the DSP composition in the biologicalsample.

Some embodiments of the invention provide methods to predict a portionof bioavailable fraction of a DSP composition. Such methods comprisecontacting a sample comprising a DSP composition with a predeterminedcapture polypeptide that is found in situ at a site where administrationand delivery of such DSP composition is contemplated, and determiningbinding of the DSP composition to the capture polypeptide. Binding by alarge fraction of the DSP composition may be indicative of a largerproportion of peptides that are therapeutically and/or physiologicallyrelevant, and tighter binding (per dissociation constant determination)may be indicative of a protective effect that extends the half-life ofthose peptides in vivo.

A further aspect of the instant invention provides methods to predict atherapeutically effective amount of a DSP composition to be administeredto a subject (e.g., a human subject) based on data obtained fromexperimental subjects. In certain embodiments, the method comprisesadministering a DSP composition to a non-human experimental mammaliansubject, determining the bioavailable portion of the dosed amount (e.g.,using a method of quantitative detection described herein), determiningfunctional read-outs, and predicting a therapeutically effective amountof the DSP composition to be delivered to the therapeutic subject basedon the data obtained for the experimental mammalian subject and acorrelation ratio between the therapeutic and experimental subjects. Forthe purposes of the instant invention, a “functional read-out” may be aphenotype or function of the subject, a phenotype or function ofcellular material derived from the subject, or the composition of one ormore fluids derived from the subject. A functional read-out mayadditionally or alternatively include a measurement of one or morebiosynthetic or metabolic components such as hormones, enzymes, serumproteins, cytokines, chemokines, growth factors, immunomodulators, andan effector or regulator of said functional read-outs. In certainembodiments, the detection step may be repeated at various regular orirregular time intervals to determine the time-course ofbioavailability, metabolism, and/or clearance after administration. Incertain embodiments, a plasma half-life of the DSP composition as agroup may be determined in this manner. In a further embodiment, ahalf-life of a species within the DSP composition may be determined inthis manner. In particular embodiments, the experimental subject is arodent, such as a mouse or rat.

Yet another aspect of the instant invention provides an efficient andeffective method of treating a patient by administering a DSPcomposition, comprising: preparing a DSP composition by synthesizingpeptides (e.g., simultaneously by using pools of amino acid monomers ateach cycle of elongation), preparing a pharmaceutically acceptableformulation of said DSP composition, administering said DSP compositionto a subject, obtaining a tissue sample from said subject, determiningthe amounts and/or concentrations of the DSP composition in said tissuesample, determining a functional read-out, correlating the amounts ofthe DSP composition to the functional read-out, and adjusting the dosageof the DSP composition to the subject to improve the functional readout.

Another aspect of the invention is a method for treating or preventingan unwanted immune response in a subject, comprising administering tothe subject a suitable dose of a DSP composition, wherein such suitabledose is determined by: (i) administering to the subject a dose of theDSP composition; (ii) removing a biological sample from the experimentalsubject; (iii) contacting the biological sample with at least onecapture polypeptide; (iv) determining a level of the capture polypeptidein the biological sample; (v) optionally repeating steps (i) through(iv) using a different dose; and (vi) comparing the level(s) against apredetermined suitable level of the DSP composition in the biologicalsample; wherein a suitable dose is the dose that results in thepredetermined suitable level of the DSP composition in said biologicalsample.

In some of the foregoing aspects and embodiments, the capturepolypeptide is labeled. In some embodiments, the capture polypeptidesare affixed to solid support. In some embodiments, the complexcomprising a capture polypeptide and one or more peptide components of aDSP composition is detected and/or isolated. In particular embodiments,the complex is detected and/or isolated by antibodies specific to thecomplex but not to the capture polypeptide or to the peptide componentof the DSP composition.

Yet another aspect of the instant invention provides a method to isolatea selected subset of the peptides that make up the DSP composition. Inparticular instances, the subset may consist of peptides having one ormore different amino acid sequences. In other instances, capturepolypeptides may be used to classify the components of the DSPcomposition based on the binding specificity.

In certain embodiments, a method for isolating peptides from a samplecomprising a DSP composition comprises: (a) contacting the sample withat least one capture polypeptide; and (b) separating peptides that bindto the capture polypeptide from the mixture. In certain suchembodiments, the capture polypeptides are affixed to a solid support. Insome embodiments, the capture polypeptides are epitope-tagged orlabeled. In some embodiments, the method further comprises separatingbound peptides from the capture polypeptides in order to isolate thepeptides. In particular embodiments, the method further comprisesdetermining the characteristics of the isolated peptides, such as aminoacid compositions of the pool of isolated peptides and/or amino acidsequences of the isolated peptides.

In certain embodiments, a method of identifying bioavailable peptides ina DSP composition in a subject comprises: (a) administering the DSPcomposition to the subject; (b) removing a tissue sample from thesubject after conducting step (a); and (c) identifying peptides in thesample that bind to at least one capture peptide.

In certain embodiments, a method of identifying a subset of peptidesthat bind to a capture polypeptide comprises preparing a DSP compositionaccording to a protocol, contacting said DSP composition with apredetermined capture polypeptide (e.g., that is desirable as in vivotarget or carrier), determining the binding of peptides within the DSPcomposition, identifying characteristics that differentiate the peptidesthat bind from peptides that do not, and preparing an improved DSPcomposition reflecting one or more of the differentiatingcharacteristics.

Another aspect of the invention is a method of improving themanufacturing process of a composition comprising a DSP composition. Insome embodiments, a DSP composition is designed based on the foregoingmethod of identifying a subset of peptides that bind to a capturepolypeptide. In some embodiments, the DSP composition is designed sothat the amino acid composition and/or the amino acid sequenceapproximates that of the subset of peptides that bound to the capturepolypeptide. In some embodiments, the DSP composition has enhancedpotency compared to a reference DSP composition, wherein the referenceDSP composition is or is substantially the same as the original DSPcomposition that was contacted with the capture polypeptide. In otherembodiments, the DSP composition has lower toxicity compared to thereference DSP composition.

In alternative embodiments, a method comprises preparing a DSPcomposition according to a protocol, formulating a compositioncomprising DSPs, determining the bioavailable amount of the DSPs in saidcomposition by detecting the level or degree of functional read-out,comparing such read-out against a standard, and adjusting the protocolor formulation of the composition to obtain a desired bioavailability.

Yet another aspect of the invention is targeting of therapeutic agentsto specific tissues by associating a DSP composition (e.g., a referenceDSP composition or an improved DSP composition generated by the methodsdisclosed herein) or a component of a DSP composition with a therapeuticagent of interest, where said DSP composition or component thereof bindsto a capture polypeptide that has tissue-specific targeting properties.Such associated agents can be administered to a patient to target theagent to a tissue associated with the corresponding capture polypeptide.

Some embodiments of this aspect of the invention provide a method fordelivering a therapeutic agent to a specific tissue in a subject, suchmethod comprising: (a) isolating a peptide tag by contacting a DSPcomposition with a tissue specific peptide and separating peptides thatbind to the tissue specific peptide from the mixture; (b) coupling thepeptide tag to a therapeutic agent; and (c) administering the conjugateto a subject. Other embodiments of the invention include a method ofpreparing such targeted therapeutic agent by step (a) and (b) of theabove described method, and a targeted therapeutic prepared thereby.

A further aspect of the instant invention is a composition useful andused in any of the methods described above. An embodiment of this aspectof the invention is a composition for detecting a DSP composition in abiological sample, comprising at least one capture polypeptide. Incertain embodiments, the capture polypeptide is selected from acomponent of normal human sera, normal non-human primate sera, normalrabbit sera, normal mouse sera, normal rat sera, normal ferret sera,normal pig sera, normal dog sera, normal horse sera, normal sheep sera,normal cow sera, a component of mammalian-derived HDL proteome, acomponent of mammalian-derived LDL proteome, complement component C3,apolipoprotein A-1 preproprotein, apolipoprotein A-II preproprotein(apolipoprotein D), complement component C4A, trypsin inhibitor,inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP),alpha-1-B-glycoprotein, alpha-1-antitrypsin, apolipoprotein A-IV,ceruloplasmin, unnamed protein product (BLAST search IDs it as a IgMheavy chain), apolipoprotein E, complement factor B, prealbumin,apolipoprotein C-III, alpha2-HS glycoprotein, apolipoprotein Jprecursor, Chain C, Immunoglobulin M, immunoglobulin lambda light chain,Coagulation factor II (thrombin), Ig kappa chain V-III (KAU coldagglutinin), apolipoprotein J precursor, Ig A1 Bur, histidine-richglycoprotein precursor, Alpha-2-HS-glycoprotein, gelsolin isoform aprecursor, inhibitor Kunitz type proteinase, unnamed protein product(NCBI Locus/Accession No. CAA28659), and Ig J-chain.

In particular embodiments the capture polypeptide may be a serum bindingprotein. In more particular embodiments, the capture polypeptide isselected from alpha-1-antitrypsin, apolipoprotein A-I,alpha-1-B-glycoprotein, apolipoprotein A-IV, apolipoprotein D, andprealbumin, or from the capture polypeptides enumerated in the paragraphimmediately preceding this paragraph, or from serum polypeptidesdisclosed herein.

Further, in any of the foregoing embodiments, the binding of peptides ina DSP composition to a capture polypeptide, such as a serum protein, maybe carried out in the presence of additional physiologically relevantcomponents. In particular embodiments, the additional component is alipid, such as cholesterol or triglycerides. In particular embodiments,the additional component is an HDL or LDL complex substantially free ofany proteinaceous component other than the capture polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of an assay used to determinebinding of a DSP composition to support-bound serum binding proteins.After the serum proteins have been identified, they are bound onsolid-support. A DSP composition, either alone or contained withinserum, is added to the support. A primary antibody against the DSPcomposition (or against the conjugate between the DSP composition andthe serum protein) is added, and binding of the primary antibody to itstarget(s) is detected by a secondary antibody and detection reagent.

FIG. 2 shows the A450 colorimetric absorbance of HRP conjugatedanti-YFAK and anti-YEAK antibodies, after the antibodies have bound totheir targets. Targets comprise complex peptides mixtures comprisingYEAK or YFAK peptides bound to serum proteins contained in (or spikedinto) normal human serum. At higher concentrations of complex peptidemixtures, the detection of conjugates by anti-YEAK or anti-YFAKantibodies is higher than lower concentrations of complex peptidemixtures. 12.5 ng/mL corresponds to a dose of approximately 2 mg in ahuman patient.

FIG. 3 shows a list of serum proteins which bind to PI-2301 or Copaxone.The origin of serum proteins is either normal mouse serum or normalhuman serum, as indicated. PI-2301 may be acetylated or non-acetylated.Binding complexes of PI-2301 or Copaxone are recognized by anti-YFAK oranti-YEAK antibodies, and detected with secondary antibodies anddetection reagents. Serum proteins are eluted from the complex andidentified. Proteins are assigned a score based on the A450 absorbanceof the detection reagent. A score of 70 corresponds to a p=0.001, ascompared to background absorbance, and is considered statisticallysignificant.

FIG. 4 shows YEAK in the serum of mice dosed IV with 4 mg/kg of YEAK orSC with 21 mg/kg using the A450 colorimetric absorbance of HRPconjugated anti-YEAK antibodies after YEAK has bound to its targetcomprised of YEAK peptides bound to serum proteins contained in normalhuman serum. The Figure shows that GA (glatiramer acetate) fragmentsreach maximum serum concentration of 1800 ng/mL at around 15 min postdosing. The estimated bioavailability of Copaxone® administered SC was12% as compared to Copaxone® administered IV. GA fraction was stilldetected in serum at 2 hours post-dosing.

FIG. 5 shows an example of the acute release of soluble factors in serumor plasma in response to YEAK administration in mice, in this caseCCL22, also known as MDC. As seen in the figure, there exists a linearcorrelation between the dose of YEAK administered SC to mice, and theobserved maximum CCL22 plasma concentration.

FIG. 6 shows peptide patterns observed by LC-MS from serum proteinseluted from YEAK fragments immobilized on a CNBr-Seph column. Peptidesequences were identified using the search engine Mascot. Briefly, YEAKfragments generated by tryptic enzyme digestion were coupled to CyanogenBromide Sepharose (CNBr-Seph) 4b, and incubated for two hours at roomtemperature with either human or mouse sera. Serum proteins bound to theYEAK fragments were eluted using a solution of 0.1M Glycine-HCL, pH 2.8,and digested with trypsin in 50% methanol/50 mM ammonium bicarbonate,dried, separated using liquid chromatography (LC), desolvated, ionized,sprayed into a mass spectrometer (MS), visualized, and identified usingthe Mascot search engine.

FIG. 7 shows an ELISA assay using methods of the instant inventiondepicted in FIG. 1 where YEAK was spiked into male and female normalhuman sera and pooled male and female normal human sera. The assaydemonstrates a linear range detecting YEAK in sera of between 1 and 100ng/ml. This assay can not be replicated using sera from mice, nor whenirrelevant controls such as anti-Keyole Limpet Hemocyanin (KLH)polyserum was used.

FIG. 8 shows an SE-HPLC profile of Copaxone® (YEAK) lots P53218, and119142 with the molecular weights demonstrated to have similar profiles.

FIG. 9 shows the two lots of Copaxone® seen in FIG. 8 used in methods ofthe instant invention depicted in FIG. 1.

FIG. 10 shows the two lots of Copaxone® used in FIGS. 8 and 9 in abioassay where the monocyte cell line RAW264.7 exposed to YEAK releasedCCL22 in a concentration dependent manner.

FIG. 11 shows using MALDI-TOF the strict linear relationship between theactual and theoretical mean molecular weights of YEAK copolymers ofdifferent defined lengths. Theoretical values were calculated bymultiplying the copolymer length in amino acids, i.e., 20, 40, 60 and80, by the average molecular weight of one theoretical amino acid plusone molecule of water. The weight of one theoretical amino acid wascalculated by using the respective mass of Y, E, A and K minus onemolecule of water lost during amino acid coupling and the amino acidratio of 1.0, 1.5, 4.5, 3.6

FIG. 12 shows the output ratios as normalized to 100 amino acids of YEAKcopolymers of different lengths manufactured by solid phase synthesisdetermined by amino acid analysis, as well as the same analysisperformed on the two lots of Copaxone® seen in FIGS. 8, 9, and 10.

Standard curves were generated, using YEAK copolymers of 20, 40, 60, and80 amino acids. For comparison, a standard curve using Copaxone was alsogenerated. FIG. 12 illustrates the relationship between size of the YEAKcopolymers and detection by the competitive ELISA-based PK assays. The20-mer YEAK copolymer has little inhibitory effect, but the standardcurve generated with the 80-mer YEAK copolymer overlays the curveobtained with Copaxone.

FIG. 13 shows an ELISA assay where the Ig fraction of rabbit polyseruminteracts strongly with Copaxone®, and demonstrates an increasingrecognition as the length of the solid phase synthesized YEAK copolymersincreases.

FIG. 14 shows an ELISA assay using a previous PK method (as described inPCT publication WO2009/075854 hereby incorporated by reference in itsentirety) with solid phase synthesized YEAK copolymers, demonstrating arelationship between the size of the YEAK copolymers and detection bythe methods of the previous assay system.

FIG. 15 shows the monocyte cell line RAW264.7 cultured with the solidphase synthesized copolymers of different sizes seen in FIGS. 12, 13,and 14 produce an increasing amount of CCL22 as the length of thecopolymer increases.

FIG. 16 shows the ability of the two lots of Copaxone® used in FIGS. 8,9, 10, and 12, and the solid phase synthesized YEAK copolymers ofdifferent lengths used in FIGS. 12,13,14, and 15 to induce ex vivoproliferation of splenocytes from mice immunized weekly for 3 weeks with2.5 mg/kg of Copaxone®. A week after the last SC administration, spleenswere collected, cell suspensions made, and the cells were cultured for 4days with various concentrations of the different copolymers. Splenocyteproliferation was determined by measuring tritiated thymidineincorporation using methods well known in the art.

DETAILED DESCRIPTION OF THE INVENTION Directed Sequence Polymer (DSP)Compositions

A DSP is a peptide having a sequence derived from a base known peptidesequence, which may be but is not limited to a native epitope associatedwith an immune response, as a starting point. A DSP has one or moreamino acid residues that differ from those of the base peptide sequence,the substitution of which is determined by a defined rule. Because ofthe semi-random diversity of a DSP composition, a large number ofpeptide sequences are present in the composition. Diversity of peptidesequences may confer increased efficacy over less diverse compositions,particularly as epitope shifting and spreading occurs. In someembodiments, a DSP composition comprising multiple DSPs is useful inmodulation of unwanted immune responses, or eliciting immune responseswhen the base peptide is weakly or undetectably immunogenic.

DSPs are designed to include a defined amino acid variation at a definedrate of occurrence of introduction of such amino acid residues at anygiven position of the sequence to the base peptide sequence. Unlike RSPssuch as Cop-1, the resulting peptides, though they may be substituted tovarying degrees, maintain their similarity to the natural sequence ofamino acid residues of a defined predetermined peptide sequence of aspecified length. Each amino acid position is subjected to change basedon a defined set of rules, such substituting amino acid selected fromchemically related amino acids, amino acids with steric similarities,phylogenic variations found in xenogeneic analogous proteins of the basepeptide, known allelic variants that do not result in dysfunction of thebase peptide, or small amino acid residues introduced to disruptsecondary structure of the peptides. In certain embodiments, the aminoacid is substituted according to the methods seen in Kosiol et al., J.Theoretical Biol., 2004, 228:97-106). Alternatively, amino acids can bechanged in accordance with the exemplary substitutions described inPCT/US2004/032598, pages 10-11.

DSPs may be prepared by solid phase peptide synthesis, and for eachcycle of the synthesis, a mixture of appropriately protected amino acidsat a defined ratio, selected for reasons described above, rather than asingle amino acid, presented for incorporation into the synthesizedpolypeptides. Which of the selected amino acids is introduced variesaccording to the mixture ratio. Thus, a DSP composition, like an RSPcomposition, is not synthesized as a single peptide, but is alwayssynthesized as part of a composition comprising multiple related DSPsbased on a common template sequence, the overall mixture of which isreproducible and consistent with the rules of synthesis that wereapplied. The result is a mixture of related therapeutically usefulproteins, which is described herein as a composition comprising“directed-sequence polymers” or “DSPs”. For a solid phase synthesisprocedure, the mixture of amino acids for a given position in thepeptide is defined by a ratio one to another. Prior to starting thesynthesis, such ratio of amino acids in the mixture available for avariant position is determined for each position along the peptide. Theresulting directed order peptide mixture comprises a multiplicity ofrelated peptide sequences. Some DSPs which may be used in the inventioninclude those described in international applications WO 2007/120834, WO2009/051797, WO 2009/128948 and US application publication US2009/0036653. These references describe methods of synthesizing DSPs,compositions comprising DSPs, therapeutic formulations of DSPs, methodsof administering DSP compositions to a subject, diseases that may betreated with DSPs, and additional therapeutically effective agents whichmay be co-administered to a subject in with the DSPs. The teachings ofall these patents, applications and publications are herein incorporatedby reference in their entirety, with particular attention to thoseportions discussing the structure, preparation, and function of theDSPs.

DSPs are designed and prepared by selecting a protein, either having noknown function, having a known or anticipated research interest, havinga known or anticipated diagnostic interest, or having a known oranticipated disease association, and selecting a portion within theprotein, which portion may be an epitope within a range ofimmunogenicity, from no known immunogenicity to being weakly immunogenicto being strongly immunogenic, or where it is known to be relevant tothe pathology of a disease. Base peptide sequences for preparing DSPcompositions may be selected from various sources. In certain instances,peptide sequences with some significance to a disease state or anadverse reaction may be identified through experimental investigation ofa relevant epitope. These sequences may include non-naturally occurringpeptide sequences that proved to be useful in treating a disease or acondition, an example found in the international patent applicationpublication WO 2006/031727, U.S. Pat. No. 6,930,168 and the relatedscientific publication by Stern et al., Proc. Nat. Acad. Sci. USA, 2005,102:1620-25.

Further, base peptide sequences that may be epitopes are empiricallydetermined by identifying candidate sequences by positional scanning ofsynthetic combinatorial peptide libraries (see, for example, D. Wilsonet al., above; R. Houghten et al., above; Hernandez et al., Eur. J.Immunol., 2004, 34:2331-41), or by making overlapping peptide sequencesof the entire protein of interest, and testing those peptides for immunereactivity using, for example, any read-out assay useful for suchpurposes, such as the HI assay, a viral challenge model, or onedescribed in Current Protocols in Immunology Edited by John E Coligan,Ada M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strober NIH,John Wiley & Sons, in an in vitro or in vivo assay system appropriatefor the disease and species the epitope is sought for. Candidatemolecules may include peptides that are modified during orpost-synthesis by, for example, sugar- and modified sugar addition suchas glycosylation and glycogenation, which may be either N or S-linked,fatty acid modification such as myristoylation, or creation of disulfidebonds.

After identifying a candidate epitope, a probable set of additionalrelated epitopes may be generated using sub-strain variants, clustervariants, drift variants, shift variants of a pathogen, via modeling andprediction algorithms described in readily available references, forexample WO 2000/042559, by aligning and analyzing the mutations,probable antibody accessible epitopes, or predicted binding of theseprobable epitopes using available prediction methods described in, forexample, WO 2005/103679, WO 2002/073193 and WO 99/45954.

In some embodiments, base peptide sequences for designing DSPs areepitopes related to an autoimmune disease selected from multiplesclerosis, systemic lupus erythematosus, type I diabetes mellitus,myasthenia gravis, rheumatoid arthritis, and pemphigus vulgaris.

In other embodiments, the base peptide sequence is an epitope relevantto the pathology of a cancer selected from leukemia, breast, skin, bone,prostate, liver, lung, brain, larynx, gallbladder, pancreas, rectum,parathyroid, thyroid, adrenal, neural, head and neck, colon, stomach,bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma,melanoma, metastatic skin carcinoma, osteosarcoma, Ewing's sarcoma,veticulum cell carcinoma, myeloma, giant cell tumor, small-cell lungtumor, islet cell tumor, lymphocytic, granulocytic, hairy-cell, adenoma,hyperplasia, medullary carcinoma, pheochromocytoma, ovarian tumor,cervical dysplasia, in situ carcinoma, neuroblastoma, retinoblastoma,soft-tissue sarcoma, kaposi's sarcoma, and osteogenic sarcoma.

In other embodiments, the base peptide sequence is an epitope relevantto the pathology of a viral infectious disease selected from AIDS, AIDSRelated Complex, Chickenpox (Varicella), Common cold, CytomegalovirusInfection, Colorado tick fever, Dengue fever, Ebola haemorrhagic fever,Hand, foot and mouth disease, Hepatitis, Herpes simplex, Herpes zoster,HPV, Influenza (Flu), Lassa fever, Measles, Marburg haemorrhagic fever,Infectious mononucleosis, Mumps, Poliomyelitis, Progressive multifocalleukencephalopathy, Rabies, Rubella, SARS, Smallpox (Variola), Viralencephalitis, Viral gastroenteritis, Viral meningitis, Viral pneumonia,West Nile disease, and Yellow fever.

In other embodiments, the base peptide sequence is an epitope relevantto the pathology of a bacterial infectious disease selected fromAnthrax, Bacterial Meningitis, Botulism, Brucellosis,Campylobacteriosis, Cat Scratch Disease, Cholera, Diphtheria, Gonorrhea,Impetigo, Legionellosis, Leprosy (Hansen's Disease), Leptospirosis,Listeriosis, Lyme disease, Melioidosis, MRSA infection, Nocardiosis,Pertussis (Whooping Cough), Plague, Pneumococcal pneumonia, Psittacosis,Q fever, Rocky Mountain Spotted Fever (RMSF), Salmonellosis, ScarletFever, Shigellosis, Syphilis, Tetanus, Trachoma, Tuberculosis,Tularemia, Typhoid Fever, Typhus (including epidemic typhus), andUrinary Tract Infections.

In other embodiments, such base peptide sequence is an epitope relevantto the pathology of a parasitic infectious disease selected fromAmoebiasis, Ascariasis, Babesiosis, Chagas Disease, Clonorchiasis,Cryptosporidiosis, Cysticercosis, Diphyllobothriasis, Dracunculiasis,Echinococcosis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis,Free-living amoebic infection, Giardiasis, Gnathostomiasis,Hymenolepiasis, Isosporiasis, Kala-azar, Leishmaniasis, Malaria,Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Pinworm Infection,Plasmodium, Scabies, Schistosomiasis, Taeniasis, Toxocariasis,Toxoplasmosis, Trichinellosis, Trichinosis, Trichuriasis,Trichomoniasis, and Trypanosomiasis (including African trypanosomiasis).

In some embodiments, the base peptide sequence is an epitope relevant tothe pathology of protein conformational disorders affecting the centraland/or peripheral nervous system, selected from Alzheimer's disease(AD), Dutch hereditary cerebral hemorrhage with amyloidosis (a.k.a.cerebrovascular amyloidosis), congophilic angiopathy; Pick's disease,progressive supranuclear palsy; familial British dementia; Parkinson'sdisease (PD), Lewy-body related diseases, multiple system atrophy,Hallervorden-Spatz disease; amyotrophic lateral sclerosis (ALS);Huntington's disease (HD); spinocerebellar ataxia; neuronal intranuclearinclusion disease; hereditary dentatorubral-pallidoluysian atrophy;prion-related diseases such as scrapie, bovine spongiformencephalopathy, variant Creutzfeldt-Jakob disease,Gerstmann-Straussler-Scheinker syndrome, kuru, fatal familial insomnia,and related disorders; hereditary cystatin c amyloid angiopathy;dementia pugilistica; and other disorders characterized by cerebralatrophy and detection of intracellular and/or extracellular fibrillaraggregates as the disorder progresses.

In a particular embodiment, the protein conformational disorder isParkinson's disease. In another embodiment, the protein conformationaldisorder is Alzheimer's disease. In another embodiment theconformational disorder is a prion-related disease. In anotherembodiment, the conformational disorder is amyotrophic lateralsclerosis. In a particular embodiment, the conformational disorder isHuntington's disease.

In other embodiments, the base peptide sequence used for the process tomanufacture the DSP composition is an epitope relevant to the pathologyof protein conformational disorders affecting multiple organs or organsother than the central nervous system, selected from: spinal and bulbarmuscular atrophy; hereditary systemic and cerebral amyloidosis,Finnish-type familial amyloidosis; senile systemic amyloidosis (a.k.a.senile cardiac amyloidosis), familial amyloid polyneuropathy; Type-2diabetes, in particular pancreatic islet amyloidosis; dialysis-relatedamyloidosis (DRA); inflammation-associated reactive systemic amyloidosis(a.k.a. AA amyloidosis); aortic medial amyloidosis; medullary carcinomaof the thyroid; hereditary renal amyloidosis; light chain associatedamyloidosis, light chain deposition disease, light chain castnephropathy, light chain cardiomyopathy; atrial amyloidosis;injection-localized amyloidosis; cystic fibrosis (CF); sickle cellanemia, and other disorders wherein fibrillogenesis is observed in theaffected organs or tissues.

Examples of natively unfolded proteins and peptides, and those suspectedto be natively unfolded, that undergo fibrillogenesis, and therefore areassociated with protein conformational disorders and may be used as thesource sequences of the base peptides for the preparation of a DSPcomposition, include: prion protein and its fragments, amyloid betaprotein and its fragments, abri protein, tau protein, alpha-synucleinand its central fragment, islet amyloid polypeptide (a.k.a. amylin),exon I of huntingtin, prothymosin alpha, amino-terminal domain ofandrogen receptor protein, ataxin-1, DRPLA protein (a.k.a. atrophin-1),and calcitonin.

Examples of globular proteins that undergo fibrillogenesis and thereforeassociated with protein conformational disorders and may be use as thesource sequences of the base peptides for the preparation of a DSPcomposition, include: cystatin c, transthyretin, beta 2 microglobulin,serum amyloid A protein and its fragments, huntingtin, immunoglobulinlight chain variable domains, insulin, lysozyme (in particular humanlysozyme), alpha lactalbumin, and monellin, ligand- and DNA-bindingdomains of androgen receptor protein, lactadherein and more specificallyits fragment (a.a. residue 245-294, a.k.a. medin), gelsolin,apolipoprotein A1, fibrinogen and its fragments, and atrial natriureticfactor.

As specific examples, in Alzheimer's disease, pathology correlatesstrongly with the presence of a 4 kDa amyloid beta (Aβ) peptide that ispart of Aβ peptide precursor (APP), cleaved by enzyme presenilin 1(PS1). Most Aβ are 40 amino acids long, and designated Aβ40, Aβ40,Aβ1-40, or, having varied amino terminal, Aβx-40. Further, studies haveindicated that the fibrillar form of Aβ1-40 stimulates the microglia,which cell type is currently thought to play an important role in thepathogenesis of Alzheimer's disease. (Jekabsone, A. et al., J.Neuroinflammation 3:24 (2006)). The peptide sequence of Aβ1-40 is shownas SEQ ID NO: 7 in Table I. On the other hand, Aβ1-42, which is a minorfraction of plaque-forming Aβ, is thought to contribute to theinitiation of the formation of fibrillar Aβ. This “long form” of thepeptide is described as SEQ ID NO: 8 in Table I. Therefore, the basepeptide sequence may be Aβ peptide, exemplified by SEQ ID NO: 8. Thebase peptide sequence may also be that of shorter peptide, i.e., Aβx-40,Aβ1-11, which has been reported in some cases to have clinicalsignificance, Aβ14-23, or Aβ16-20. Tjernberg, L. O. et al., Biochem. J.366:343-351 (2002).

TABLE I Examples of epitopes Source/ SEQ Original Residue ID RelevancePeptide Sequence Protein Number Ref NO: Neuro- DAEFRHDSGYEVHHQKLVFFAAmyloid beta  1-40 54  7 degeneration EDVGSNKGAIIGLMVGGVVDAEFRHDSGYEVHHQKLVFFA Amyloid beta  1-42 55  8 EDVGSNKGAIIGLMVGGVVIAMGKGEEGYPQEGILEDMPVDP Mouse alpha 100-140 56  9 GSEAYEMPSEEGYQDYEEAsynuclein DNEAYEMPSEEGYQDYE Human alpha 121-137 57 10 synucleinMATLEKLMKAFESLKSF Huntingtin  1-17 58 11 Dialysis- IQRTPKIQVYSRHPAENGKSBeta-2 21-40 59 12 related microglobulin amyloidosis Reference: Näslund,J. et al., Proc. Nat. Acad. Sci. USA, 91: 8378-8382 (1994) 54 Gandy, S.,J. Clin. Invest. 115(5): 1121-1129 (2005) 55 Benner. E. J. et al., PLoSONE 3(1): e1376 (2008) 56 Campion. D. et al. “The NACP/synuclein gene:chromosomal assignment and screening for alterations in Alzheimerdisease” Genomics 26 (2), 254-257 (1995) 57 Lecerf, J.-M. et al, ProcNatl Acad Sci USA. 98(8): 4764-4769 (2001) 58 Kozhukh, G V et al, JBC,Vol. 277, No. 2, Issue of January 11, pp. 1310- 1315, 2002. 59

DSPs can also be used to treat Parkinson's Disease (PD). PD is adegenerative neurological disorder currently without a cure affecting1-2% of the individuals over 50 years of age. The neuropathologicalhallmarks are characterized by progressive loss of neuromelanincontaining dopaminergic neurons in the substantia nigra pars compacta(SNpc) with the presence of eosinophillic, intracytoplamic,proteinaceous inclusions termed Lewy Bodies (LB). α-Synuclein is themost abundant protein in Lewy Bodies, and appears to be an importantmediator, perhaps even a causal factor, of toxicity in PD. Thus,reduction of toxic α-Synuclein is thought to be beneficial to PDpatients. The sequence of one such mouse α-Synuclein peptide, derivedfrom the C-terminal region of the full length protein, is shown as SEQID NO: 9 in Table I. (Benner, E. J. et al., PLoS ONE 3(1): e1376(2008)). Further, elimination or sequestration of nitrated α-Synucleinand fragments thereof, appear to have favorable effects on the patientssuffering from PD. Therapeutically effective antibodies are said to bedirected at the nitrated α-Synuclein but not native. Therefore, the basepeptide sequence may be, for example, SEQ ID NO: 9. In otherembodiments, the base peptide sequence may be a fragment comprisingamino acids 121-137 of human α-Synuclein (DNEAYEMPSEEGYQDYE) (SEQ ID NO:10). In yet other embodiments, the α-Synuclein fragment (121-137)sequence is substituted at positions 121 and 122 in different species,tri-nitrated at each Y (tyrosine) position, and/or phosphorylated atS115.

DSPs may also be derived from base peptide sequence relevant toprion-diseases. SEQ ID NO: 13 (AAH22532) is human prion proteinsequence. A relevant peptide is selected from partial sequences of SEQID NO: 13. Various species' prion sequences are disclosed by Harmeyer,S. et al., J Gen Virol. 79(Pt 4):937-45 (1998), the entirety of which isincorporated herein by reference. The amino acid variations by speciescan be used to design the substituting amino acids.

A base peptide sequence may also be derived from superoxide dismutase I(SOD1). SOD1 mutation is known to have a causal relationship with thepathology of some forms of familial ALS. It has been reported that theantisera raised against a mutant form of SOD1, human G93A SOD1recombinant protein, had protective effect on a mouse model of ALScarrying G37R mutant SOD1 (line 29), which overexpress human SOD1protein by 4-fold higher than endogenous mouse SOD1. Urushitani, M. etal., Proc. Nat. Acad. Sci. USA, 104(7): 2495-2500 (2007). An example ofSOD1 protein sequence is SEQ ID NO: 14 (CAG46542). Therefore, a basepeptide sequence may be a partial sequence of SEQ ID NO: 14.

Misfolded protein also plays a role in Huntington's disease, a geneticdisorder caused by the pathological expansion of a polyglutamine (polyQ)tract in the huntingtin (htt) protein (SEQ ID NO: 15, human huntingtin),resulting in neurodegeneration and premature death of the afflictedindividual. A single-chain antibody that binds to an epitope formed bythe N-terminal 17 amino acids of htt (Lecerf, J. -M. et al., Proc NatlAcad Sci USA. 98(8): 4764-4769 (2001) SEQ ID NO:11) has been shown toreduce symptoms in a Drosophila model of Huntington's disease.(Wolfgang, W. J. et al., Proc Natl Acad Sci USA. 102(32): 11563-11568(2005)) Therefore, a base peptide sequence may be SEQ ID NO: 11.

DSP compositions may also be used to treat Dialysis-related Amyloidosis(DRA). DRA may be caused by different forms of blood filtration, such ashaemodialysis, hemofiltration, or Continuous Ambulatory PeritonealDialysis (CAPD). DRA has an incidence of greater than 95% of patients ondialysis for more than 15 years with beta-2-microglobulin (B2M, SEQ IDNO:13) amyloidosis being prevalent and predictably increasing over time.Conformational isomers of B2M have been observed in a clinical setting(Uji et al., Nephron Clin Pract 2009;111:c173-c181). B2M is part of thehuman leukocyte antigen (HLA) class I molecule, and has a prominentbeta-pleated structure characteristic of amyloid fibrils. B2M is knownto circulate as an unbound monomer distributed in the extracellularspace. B2M undergoes fibrillogenesis to form amyloid deposits in avariety of tissues. This deposition causes renal failure, which causesan increase in synthesis and release of B2M, exacerbating the condition.Thus, in an embodiment of the invention, a protein the base sequence ofwhich is used for preparation of a DSP composition is beta 2microglobulin (SEQ ID NO: 16) and fragments thereof. An exemplaryfragment of B2M may be that spanning amino acid residues 21-40, SEQ IDNO: 12 in Table I, useful as a base peptide for DRA.

In other embodiments, the base peptide sequence is a partial sequence ofa protein selected from: osteopontin, an HLA protein, myelinoligodendrite glycoprotein, myelin basic protein (MBP), proteolipidprotein, and myelin associated glycoproteins, S100Beta, heat shockprotein alpha, beta crystallin, myelin-associated oligodendrocytic basicprotein (MOBP), 2′,3′ cyclic nucleotide 3′-phosphodiesterase, hsp60,hsp70, Ro60, La, SmD, and 70-kDa U1RNP, glutamic acid decarboxylase(GAD65), insulinoma-antigen 2 (IA-2), insulin, acetylcholine receptor(AChR) α-subunit and muscle-specific receptor tyrosine kinase (MuSK),type II collagen, desmoglein 1 (Dsg1), desmoglein 3 (Dsg3), G-proteincoupled receptors (GPCR), inflammatory related proteins, allergicrelated proteins, interleukins and their receptors, chemokines and theirreceptors, chaperones and their receptors. In other embodiments, thebase peptide sequence is derived from CD20, vascular endothelial growthfactor (VEGF), CD52, epidermal growth factor receptor (EGFR+), CD33,HER2; non-oncology related proteins, e.g., TNF alpha, CD25 orimmunoglobulin E, for immunosuppression, CD11a, alpha4-beta1 integrin;infectious disease related beta chemokine receptor CCR5, RSVgpP.

Alternatively, a base peptide sequence may be created from adiscontinuous epitope, that is, selecting the amino acids that make upthe epitope, combining the amino acids into a linear peptide toperforming directed permutations to create the DSP composition.

Yet other embodiments of the instant invention comprise selecting two ormore proteins of interest, from which two or more epitopes are selectedwith at least one epitope deriving from each protein of interest, andcombining the epitopes into a linear sequence to performing directedpermutations to create the DSP composition.

In still further embodiments, a base sequence to prepare DSPs is takenfrom the group of proteins comprising: a protein known only ascontaining a domain having a primary, secondary tertiary or quaternarystructural attribute, such as beta pleated sheet or alpha helices, aprotein known only as containing a domain having a certain activity,such as serotonin binding, a protein known only as having a knownorigin, a protein known only as belonging to a specific cellularcompartment such as the nucleus or cytoplasm, a protein known only ashaving a cellular function, such as a cellular process producing aspecific protein of interest, a protein known only as having anantioxidant activity or a metabolic activity, or a biosynthesisactivity, or a catabolic activity, or a kinase activity, or atransferase activity, or a lyase activity, or a ligase activity, or asignal transduction activity or a binding activity, or a motilityactivity, or a membrane fusion activity, or a cellular communicationactivity, or a biological process regulation activity, response tostimulus activity, a cellular death related activity, a T cellactivation related activity, a B cell activation related activity, anAPC activation related activity, an inflammatory immune response relatedactivity, an allergic response related activity, an infectious diseaseresponse related activity, a transporter activity, a channel activity, asecretion activity, a pathogenic activity, and a cytoskeletonorganization activity.

DSP compositions can be classified according to their preferentialbinding targets and their physiological functions, which derive directlyfrom the amino acid composition and their ratios. Any available methodcan be used to ascertain whether a DSP composition binds to a candidateor known target proteins. For example, the polypeptide can be labeledwith a reporter molecule (such as a radionuclide or biotin), mixed witha crude or pure preparation of a target protein and binding is detectedif the reporter molecule adheres to the target protein after removal ofthe unbound polypeptide.

In particular embodiments, DSP compositions useful for the presentinvention bind to one or more DQ isotypes with an average K_(d) of 1 μMor less, and more preferably an average K_(d) less than 100 nM, 10 nM oreven less than 1 nM. Another way to identify preferred DSPs is based onthe measure of a DSP composition to displace another in competitivebinding assays, using assays akin to those described in Sidney et al.,2002, J. Immunol. 169:5098, which is expressed as an IC₅₀ value. In someembodiments, DSPs of the present invention have IC₅₀'s less than 1 μM,more preferably less than 500 nM, and even more less than 100 nM.

In the methods herein, DSPs can be substituted with peptide pools,peptide libraries, or pools of altered peptide ligands (APLs). Like DSPcompositions, APL compositions comprise a mixture of relatedpolypeptides. APLs are defined as a series of peptides each of which hasa small number of amino acid changes from a starting sequence ofinterest, such as that of a native immunogenic peptide ligand. Variantpeptides with such altered amino acid sequences may be pooled to preparea composition having the advantages of a heterogeneous peptide mixture.Fairchild et al., Curr. Topics Peptide & Protein Res. 2004, 6:237-44.Each APL would have a defined sequence, but the composition may be amixture of APLs with more than one sequence. In some embodiments, poolsof peptides or APLs or peptide libraries which may be used in theinstant invention include those described in U.S. Pat. No. 7,118,874.

Pharmacokinetic Methods

In some embodiments, the absorption and distribution of DSP compositionsmay be determined. The rate at which a DSP composition effects a changeand the persistence of the effect, as well as chemical alterations tothe composition of the DSP composition may also be determined.

Different DSP compositions will persist for different lengths of time inthe serum and other biological fluids than other mixtures. In someinstances, the administered peptides are sequestered by or bound to somein vivo component in situ, the result of which is longer half-life inthat environment, with or without enhancement in bioavailability. Incertain embodiment, the environment is blood plasma or lymph. In analternative embodiment, the environment is spinal or cerebral fluid. Inyet other embodiments, the environment is any tissue or organ locale towhich peptides from DSP compositions are delivered.

Identification of Physiological Polypeptides and Proteins that BindAmino Acid Polymers from DSP Compositions

One aspect of the present invention is identification of a capturepolypeptide that binds a DSP composition. The term “capture polypeptide”is used herein to mean any polypeptide, protein, protein fragment,proteolipid, or other molecule containing proteinaceous material, foundin normal tissues and organs. It may be a single polypeptide or aprotein comprising multiple polypeptides and/or subunits, or a complexcomprising a protein associated (covalently or non-covalently) withother materials such as lipids, which may further have definedstructures that are desirable or necessary for the capture polypeptideto bind a DSP composition. Often a capture polypeptide is not transient,i.e., there is a base, stable amount that is found at all times,regardless of whether there is an induced or enhanced presencetransiently. Preferably, a capture polypeptide is a protein. Morepreferably, a capture polypeptide is a protein found in a biologicalfluid, such as a serum protein.

Some embodiments of this aspect of the invention are methods ofidentifying a capture polypeptide that binds to peptides that compose aDSP composition, wherein the methods comprise: contacting a samplecontaining an amount of the DSP composition with a normal tissue sample;and detecting binding of the peptides of the DSP composition to anycomponent of the normal tissue sample. In certain embodiments, thepeptides of DSP composition are immobilized either on a resin (throughcovalent bond by reacting the peptides with activated resin) or on asolid substrate such as polystyrene. For example, a tissue sample may becontacted with the immobilized peptides and incubated, washed to removenon-specific binding, and the materials bound to the peptides that werein the tissue sample identified. The bound materials may be identifiedby any suitable method, such as by subjecting the materials to a panelof specific antibodies; microsequencing of materials if such materialsare suspected to be polypeptides or nucleotides; tryptic digestionfollowed by liquid chromatography coupled with tandem mass spectrometry(LC-MS/MS) subjecting such materials to specific dyes if such materialsare suspected to be polysaccharides; or any analytical method withsufficiently high sensitivity.

As a non-limiting example of the above described identification, a DSPcomposition may be used in a direct ELISA assay to identify serumproteins that bind to the DSP composition using a protocol like that inExample 1. Table II below lists serum proteins experimentally shown tobind to RSPs, YEAK and/or YFAK peptides, in normal human serum. It hasbeen observed that YEAK and YFAK peptides have different bindingspecificities; conversely, serum proteins can be said to bind YEAK andYFAK peptides with different specificities. Tables III and IV list serumproteins which associate with HDL and LDL, respectively. Any serumproteins may bind to the DSPs described herein by varying affinities andselectivities.

Once a capture polypeptide that binds DSPs is identified, thespecificity of the binding against similar peptides or againstcompletely random peptides may be determined. The identified andcharacterized capture polypeptide (either the same molecules actuallyidentified or like molecules obtained from a different source) then inturn may be used to quantitatively analyze the DSP compositions that itwas found to bind.

Serum Proteins

In some embodiments, binding of DSP compositions to serum proteinsconstitutes an important aspect of their biological activity. Thebinding of DSP compositions to serum proteins may facilitate theirtissue distribution and capture by antigen-presenting cells such asmonocytes and macrophages. As stated above, binding of peptides to serumproteins may protect them from degradation and/or turnover. In ananalogous experiment involving RSPs, PI-2301 (plovamer, a YFAK randomsequence polymer) and Cop-1 (glatiramer acetate, a YEAK RSP) can bedetected in serum of various species, including man, several hours aftersubcutaneous administration, whereas a control RSP disappeared fromserum after a short time (US App. Pub. 2009-027496). Copolymer 1 (Cop-1)is also referred to as glatiramer acetate. Cop-1 has been approved inseveral countries for the treatment of multiple sclerosis (MS) under thetrade name, COPAXONE™ (trademark of Teva Pharmaceuticals Ltd., PetahTikva, Israel). Molecular weight ranges and processes for making apreferred form of Cop-1 are described in U.S. Pat. No. 5,800,808.

Accordingly, serum proteins may be used to capture and/or identify oneor more peptides from a DSP composition. As a whole, DSP compositionscontain a large number, even billions, of individual peptides, of whichone or more sub-fractions may be responsible for the serum proteinbinding properties, while other sub-fractions are not. This isespecially true for mixtures made by solution phase peptide synthesis,where different lots of DSP compositions may contain variations in thepercentage of peptides capable of binding serum proteins. For example,it may be important to monitor DSP compositions in serum to demonstratebioequivalence among different lots in order demonstrate that the serumprotein-binding fractions are equivalent quantitatively andqualitatively across different lots of DSP compositions.

Serum proteins may be used in vitro to select and/or characterizebinding partners from a DSP composition. Serum proteins may also be usedin vivo to select, measure, and/or otherwise characterize peptides whichbind the serum proteins, thus providing a means for distinguishingspecific peptides or subsets of peptides on the basis of their bindingto serum proteins and/or their persistence in vivo. Specificcharacteristics of peptides that bind to serum proteins may comprisespecific amino acid sequences, ratios of amino acids in the mixture,structures, unique motifs, configuration of charged residues.

TABLE II Examples of serum proteins experimentally shown to bind to YEAKand YFAK peptides in human serum: NCBI locus/ Protein Accession No.alpha-1-antitrypsin (SEQ ID NO: 1) AAA51546 (CAJ15161)alpha-1-B-glycoprotein (SEQ ID NO: 3) OMHU1B alpha2-HS glycoproteinBAA22651 Alpha-2-HS-glycoprotein P02765 apolipoprotein A-1 preproprotein(SEQ ID NO: 4) AAA51747 Apolipoprotein A-I (SEQ ID NO: 2) Q9Z2L4(AAS68227) apolipoprotein A-II preproprotein (apolipoprotein D)NP_001634 (AAB32200) apolipoprotein A-IV AAA51744 apolipoprotein C-IIIAAB59372 apolipoprotein D (SEQ ID NO: 5) AAB35919 apolipoprotein EAAB59518 apolipoprotein J precursor AAA51765 ceruloplasmin AAA51975Chain C, Immunoglobulin M 2RCJ_C Coagulation factor II (thrombin) 3F68_Hcomplement component 3 NP_058690 complement component C3 AAA85332complement component C4A AAA51855 complement factor B AAA16820 gelsolinisoform a precursor NP_000168 histidine-rich glycoprotein precursorNP_000403 Ig A1 Bur 763134A Ig J-chain AAA58902 Ig kappa chain V-III(KAU cold agglutinin) A23746 immunoglobulin lambda light chain CAA40939inhibitor, Kunitz type proteinase 0511271A inter-alpha-trypsin inhibitorfamily heavy chain-related BAA07602 protein (IHRP) Inter-alpha-trypsininhibitor heavy chain H1 Q61702 Inter-alpha-trypsin inhibitor heavychain H2 Q61703 lumican AAB35361 Prealbumin (SEQ ID NO: 6) BAA00059trypsin inhibitor CAA30160 unnamed protein product (putative IgM heavychain) CAA34971 unnamed protein product (putative vitronectin) CAA28659vitronectin AAA40558

TABLE III serum proteins associated with HDL Proteins Accession No. ApoA-I P02647 Apo A-II P02652 Apo A-IV P06727 Apo C-II P02655 Apo C-IIIP02656 Apo D P05090 Apo E P02649 Apo J P10909 Apo L1 O14791 Apo M gi13645390 LPL gi 3293305 CETP P11597 C-RP P02741 Ceroplasmin gi 13645230Complement component 3 gi 13649325, Haptoglobin gi 1212947, P00738 SAAP35542 SAP P02743 Transthyretin P02766 Transferrin gi 4557871, P02787PON P27169 Complement component 1 inhibitor P05155 Macrophagestimulating factor 1 gi 10337615 Lymphocyte antigen gi 553540 Meningiomaexpressed antigen 5 gi 11024698 HLA-A protein gi 13620230 NOTCH1 gi11275980 Sialic acid binding Ig-like lectin 5 gi 13633818 C-type lectinsuper family member1 gi 5031637 H factor 1 (complement) gi 4504375Complement component 3 gi 13649325, Insulinoma-associated protein I A-6gi 14211925 Latent transforming growth factor beta gi 3327808 LTBP-2 gi1272664 Growth arrest-specific gene-6 gi 4557617 Receptors ryanodinereceptor 2 gi 13638463 POU 5 domain protein gi 12382246 Plasmakallikrein B1 gi 11436257 TFPI P10646/P48307 Unnamed protein product gi10435007 Unknown protein gi 12653035 Unknown protein gi 12802992KIAA1095 gi 5689527 KIAA1730protein gi 12698005 KIAA0675 gene product gi13643803 CIP-interacting zing finger protein gi 12643326 dj675G8.1(novelzinc finger protein) gi 11137825 dj733D15.1 gi 3702137 TAT-interactiveprotein, 72-kDa gi 1427566 dj758N20.1 (protein kinase) gi 11493357Protein tyrosine phosphatase gi 13645209 Hypothetical proteindj1057B20.2 gi 11034845 Desmocollin gi 13435361 Coagulation factorVIII-associated protein gi 13652210 IgG gi 10334541, P99007 HSA gi178345, P02728 α-1β-glycoprotein P04217

TABLE IV serum proteins associated with LDL Proteins apoE (fiveisoforms) apoL-I (seven isoforms) apoC-IV (three isoforms) apoA-IVapoA-I apoM apoC-III b-actin fibrinogen-g (two isoforms) albumin (threeisoforms) Prenylcysteine lyase (two isoforms)

Binding Between Serum Proteins and DSP Compositions

Without wishing to be bound by theory, mechanistically, binding ofpeptides within DSP compositions, to the serum proteins such aslipoproteins found associated with HDL and LDL might facilitate theircapture by monocytes through receptors such as SR-BI or ABCA1. Thisbinding may induce activation of monocytes and their differentiationinto anti-inflammatory cells.

A serum protein may bind to a DSP composition as a part of a cholesterolcomplex such as an HDL or LDL complex, and/or in conjunction with otherproteins and polypeptides (any of which individually may also functionas a capture polypeptide) that are found in association with the serumprotein under physiological conditions. Thus, the methods of theinvention contemplate having additional components found in the serumwhen binding DSP composition to a serum protein.

Detection of a DSP Composition in a Biological Sample Determination ofBioavailability

One aspect of the instant invention is a method for detecting thepresence of a DSP composition in a biological sample, comprising:contacting the biological sample with at least one capture polypeptide;and detecting the presence or absence of binding of the capturepolypeptide to the DSP composition, wherein the presence of bindingindicates the presence of peptide components of the DSP composition inthe biological sample. Further, such method can be extended to measurethe amount or concentration of a DSP composition in a sample.

In some embodiments, the presence of a DSP composition may be detectedin a biological sample by contacting the biological sample with at leastone capture polypeptide (e.g., comprising a peptide selected fromalpha-1-antitrypsin, apolipoprotein A-I, alpha-1-B-glycoprotein,apolipoprotein A-IV, apolipoprotein D, and prealbumin); and detectingthe presence or absence of binding of the capture polypeptide to the DSPcomposition. In this assay, the presence of binding indicates thepresence of DSPs in the biological sample. Further, the inventionprovides methods for determining an amount of a DSP composition in abiological sample, by contacting the biological sample with at least onecapture polypeptide (e.g., comprising a peptide selected fromalpha-1-antitrypsin, apolipoprotein A-I, alpha-1-B-glycoprotein,apolipoprotein A-IV, apolipoprotein D, and prealbumin); and quantifyinga level of binding of the capture polypeptide to the DSP composition.

Other embodiments of the invention provide methods of determining thebioavailability of a DSP composition in a subject, comprisingadministering to a subject a dose of a composition comprising the DSPcomposition; removing a biological sample from the subject; andcontacting the biological sample with at least one capture polypeptide(e.g., comprising a peptide selected from alpha-1-antitrypsin,apolipoprotein A-I, alpha-1-B-glycoprotein, apolipoprotein A-IV,apolipoprotein D, and prealbumin). It is contemplated that the peptidesof DSP compositions are extensively bound to a capture polypeptide invivo. Nevertheless, for further characterization, antibodies specificagainst the complexes comprising peptides of a DSP composition and acapture peptide, but not each of those singly, may be used for detectionof the bioavailable DSP composition.

Improvement of Dosage and Methods of Administration

Another aspect of the instant invention provides methods ofadministering DSP compositions to a mammalian subject, in an amountdetermined based on the bioavailable portion of the dosed amount asdetermined by the method described above or other methods describedherein. In certain embodiments, the method further comprises including acontrol sample, performing a pharmacodynamic test to determine changesof physiological markers, such as hormones, enzymes, serum proteins,cytokines, immunomodulators, or an effector or regulator of any of thesefunctional proteins, between the control sample and test samples bycomparing the two results, and determining the dosage effective toinduce the desired changes in a pharmacodynamic parameter. In certainembodiments, behavioral changes, subjective changes as reported by asubject such as amelioration of pain or a symptom of a disease, or otherevidence of indirect effects are observed. In certain embodiments, saidmammalian subject is a rodent, such as a mouse or rat. In otherembodiments, said subject is human.

More generally, a method for treating or preventing an unwanted immuneresponse in a subject may comprise providing a DSP composition;administering the DSP composition to a test subject; removing abiological sample from the test subject; contacting the biologicalsample with at least one capture polypeptide (e.g., comprising a peptidesequence selected from alpha-1-antitrypsin, apolipoprotein A-I,alpha-1-B-glycoprotein, apolipoprotein A-IV, apolipoprotein D, andprealbumin); separating DSPs that bind to the capture polypeptide fromthe mixture; determining characteristics of the separated DSPs;preparing a set of DSPs with the characteristics of the separated DSPs,and administering the prepared set of DSPs to a subject.

In these methods, DSP compositions may be administered to a subject morethan once. DSP compositions may be administered to the subject atintervals of, for example, 1, 2, 3, 4, 6, 12, 18, 24, 36, 48, or 72hours.

Thus, some embodiments of the invention are methods of administering asuitable dose of a DSP composition to a subject in need thereof, whereinthe suitable dose is determined by administering to the subject a firstdose of the DSP composition; removing a biological sample from thesubject; contacting the biological sample with at least one capturepolypeptide (e.g., comprising a peptide selected fromalpha-1-antitrypsin, apolipoprotein A-I, alpha-1-B-glycoprotein,apolipoprotein A-IV, apolipoprotein D, and prealbumin); determining alevel of the capture polypeptide in the biological sample; optionallyrepeating the previous steps using a second different dose; andcomparing the levels to a predetermined suitable level of the DSPcomposition in the biological sample. Under these conditions, a suitabledose is the dose that results in the predetermined suitable level of theDSP composition in the biological sample. A suitable level of a DSPcomposition in a biological sample is a level at which a desirablefunctional read-out, or surrogate marker change, is obtained. Afunctional read-out can be the phenotype or function of the subject, thephenotype or function of cellular material derived from the subject, orthe composition of fluids derived from the subject. In a particularembodiment, the detection step is repeated after certain time intervalsto determine the time-course of bioavailability after administration. Incertain embodiment, a half-life of the DSP composition as a group isdetermined from such time course. Examples for functional readouts ofimmune response enhancement or sequestering are: increase or detectionof TNFα, IL-6, CXCL1, CXCL2, and IL-12p70 as indicators of undesiredimmune stimulation, and increase or detection of II-Ira, CXCL13, andCCL22 as indicators of desirable positive changes. Changes in thesemarkers are easily determined by skills and materials known and readilyavailable in the art.

Certain embodiments of the invention facilitate the comparison ofeffective doses across species. Comparison of effective doses in humanand experimental animals such as mice or rats is made difficult not onlyby the body size difference and the difference in general metabolism,but also because it has been observed that bioavailability of a drugdiffers between animal species. It is an aspect of the present inventionthat the bioavailability of DSP compositions is correlated partly by thebinding of the component peptides to serum proteins, which may allow forlonger half-life and certain tissue distribution. Thus, some embodimentsof the invention are methods of determining a suitable dosage of a DSPcomposition in a subject, such methods comprising determining a firstsuitable dosage of the DSP composition in an experimental animal model,wherein the first suitable dosage is such dosage that gives a favorableread-out and that corresponds to a level of DSP composition bound to aserum protein in vivo, and determining a second suitable dosage of theDSP composition in the subject by dosing the subject so that the levelof DSP composition bound to the serum protein in vivo in the subject issimilar or identical to the level achieved by administering the firstsuitable dosage to the experimental animal.

In particular embodiments, administration of a DSP composition may beenhanced using the methods of present invention. One method comprisesadministering to the subject a suitable dose of a DSP composition,wherein such suitable dose is determined by administering to the subjecta dose of the DSP composition; removing a biological sample from theexperimental subject; contacting the biological sample with at least onecapture polypeptide (e.g., selected from alpha-1-antitrypsin,apolipoprotein A-I, alpha-1-B-glycoprotein, apolipoprotein A-IV,apolipoprotein D, and prealbumin); determining a level of the capturepolypeptide in the biological sample; optionally repeating all previoussteps, and comparing the level(s) against a predetermined suitable levelof the DSP composition in the biological sample. A suitable dosage isdetermined as described above, based on favorable readouts.

Peptides may be labeled by any suitable means, such as affixingfluorescent moieties, radioactive labels, forming chemical conjugates,biotinylation, adding epitope tags, or any other moiety that facilitatesdetection. Serum proteins acting as detector polypeptides as describedabove may be affixed to a solid support. After serum proteins have boundto one or more peptides from the DSP composition, the bound complexcomprising the capture polypeptide bound to the DSP composition may beisolated.

Methods for isolating bound complexes may include immunoprecipitation,ELISA, immunodetection, or detection of the label the capturepolypeptides. Detecting binding of the capture polypeptide to the DSPcomposition may be performed with antibodies to the capture polypeptide,antibodies to the DSP composition, or antibodies that have beengenerated to recognize the bound complex.

DSP compositions may be administered subcutaneously, intramuscularly,intravenously, intranasally, or through any orifice or mucous membrane.

In some embodiments, a composition for detecting a DSP composition in abiological sample may comprise at least one capture polypeptidecomprising a peptide selected from alpha-1-antitrypsin, apolipoproteinA-I, alpha-1-B-glycoprotein, apolipoprotein A-IV, apolipoprotein D, andprealbumin.

Selection of Specific Peptides From Within a DSP Composition

An aspect of the present invention is its use in identifying and/orisolating peptides or a subset of peptides from a DSP composition.Although one advantageous feature of the DSP compositions compared to asingle-species or oligo-specific peptide samples is its heterogeneity,it is conceivable that a subset of the peptides that compose the mixtureis more effective than another subset, or that a subset is in factundesirable. Thus, the present invention provides methods foridentifying and/or isolating peptides from a sample comprising a DSPcomposition based on the peptides' affinity to certain capturepolypeptides. In particular instances, the subset may comprise peptideshaving one or more different amino acid sequences. In other instances,capture polypeptides may be used to classify the components of the DSPcomposition based on the binding specificity.

In some embodiments, a method of identifying a subset of peptides thatbind to a capture polypeptide comprises preparing a DSP compositionaccording to a protocol, contacting said DSP composition with apredetermined capture polypeptide (e.g., that is desirable as in vivotarget or carrier), determining the binding of peptides within the DSPcomposition, identifying characteristics that differentiate the peptidesthat bind from peptides that do not, and preparing an improved DSPcomposition reflecting one or more of the differentiatingcharacteristics.

In certain embodiments, a sample containing a DSP composition iscontacted with a capture polypeptide, and the peptides that compose theDSP composition that bind to the capture polypeptide are isolated andidentified. In certain embodiments, a DSP composition is contacted withat least one serum protein which acts as a capture polypeptide. In moreparticular embodiments, such serum protein is selected fromalpha-1-antitrypsin, apolipoprotein A-I, alpha-1-B-glycoprotein,apolipoprotein A-IV, apolipoprotein D, and prealbumin capturepolypeptide.

The capture polypeptide may be immobilized on a solid support, and/ormay be labeled by methods known in the art. Immobilization and labelingmay be used in further steps of separating bound peptides from thecapture polypeptides, and/or determining characteristics of isolatedpeptides. Such characteristics may include the amino acid sequence of abound peptide, relative ratios of amino acids in bound peptides,configuration or disposition of charged residues in the sequence, thestructure of the peptide, charge, or any other suitable characteristic.

The binding between DSP compositions and serum proteins may also be usedfor identifying bioavailable peptides in a DSP composition, such as abiological sample collected from a subject. Here, the DSP compositionmay be administered to the subject at a first time; and then, at asecond time after administration, a tissue sample may be removed fromthe patient. In the tissue sample, peptides in the sample that bind toat least one capture polypeptide, e.g., comprising a peptide selectedfrom alpha-1-antitrypsin, apolipoprotein A-I, alpha-1-B-glycoprotein,apolipoprotein A-IV, apolipoprotein D, and prealbumin, may beidentified.

Improved Preparation of DSP Compositions

Another aspect of the invention is a method of improving themanufacturing process of a composition comprising a DSP composition. Insome embodiments, a DSP composition is designed based on the foregoingmethod of identifying a subset of peptides that bind to a capturepolypeptide. In some embodiments, the DSP composition is designed sothat the amino acid composition and/or the amino acid sequenceapproximates that of the subset of peptides that bound to the capturepolypeptide.

In certain embodiments, a method for producing a DSP composition havingreduced toxicity may comprises contacting the DSP composition with atleast one capture polypeptide (e.g., comprising a peptide selected fromalpha-1-antitrypsin, apolipoprotein A-I, alpha-1-B-glycoprotein,apolipoprotein A-IV, apolipoprotein D, and prealbumin); separatingpeptides that bind to the capture polypeptide from the mixture;determining characteristics of the separated peptides; and preparing aset of peptides with the characteristics of the separated peptides.

Similarly, a method for producing a DSP composition having enhancedpotency may comprise contacting the DSP composition with at least onecapture polypeptide comprising a peptide (e.g., selected fromalpha-1-antitrypsin, apolipoprotein A-I, alpha-1-B-glycoprotein,apolipoprotein A-IV, apolipoprotein D, and prealbumin); and separatingpeptides that bind to the capture polypeptide from the mixture;determining characteristics of the separated peptides; and preparing aset of peptides with the characteristics of the separated peptides.

In some embodiments, a desirable subset of a DSP composition may beobtained by using immobilized capture polypeptides in a preparatoryscale. A DSP composition is prepared as previously contemplated anddescribed, and contacted with immobilized capture polypeptides relevantto a desired improvement. Unbound peptides are removed by washing thesample, and bound portion of the DSP composition is eluted usingappropriate dissociation condition, such as varied pH, saltconcentration, or addition of organic solvents. The pooled bound portionis treated appropriately to concentrate and to remove therapeuticallyundesirable components, e.g. organic solvent, by evaporation or byfurther purification through appropriate chromatographic orcrystallization or other purification methods. The subset of the DSPcomposition thus prepared is used as therapeutic agents.

Further, this aspect of the invention may be combined with theabove-described improvements in dosage and administration. Whenbetter-tailored DSP compositions are prepared, it is anticipated thatthe dosage and mode of administration may be adjusted accordingly.Therefore, in alternative embodiments, a method comprises preparing aDSP composition according to a protocol, formulating a compositioncomprising the DSP composition, determining the bioavailable amount ofthe DSP composition in said composition by detecting the level or degreeof functional read-out, comparing such read-out against a standard, andadjusting the protocol or formulation of the composition to obtain adesired bioavailability.

Tissue-specific Targeting of Therapeutic Agents

Another potential use of the relationship between DSP compositions andserum proteins is tissue-specific targeting of therapeutic agents. Inone embodiment, a method for preparing a therapeutic agent to a targettissue in a subject may comprise providing a DSP composition; andcoupling a therapeutic agent to the DSP composition to form a conjugate.

Thus, some embodiments of the invention are methods for delivering atherapeutic agent to a specific tissue in a subject by isolating apeptide tag by contacting a DSP composition with a tissue-specificpeptide (e.g., comprising a peptide selected from alpha-1-antitrypsin,apolipoprotein A-I, alpha-1-B-glycoprotein, apolipoprotein A-IV,apolipoprotein D, and prealbumin); and separating peptides that bind tothe tissue-specific peptide from the mixture; coupling the peptide tagto a therapeutic agent; and (c) administering the conjugate to asubject.

Other embodiments of the invention include a method of preparing aconjugate comprising a therapeutic agent coupled to a peptide tag, andthe resulting conjugates themselves. Such a peptide may be isolated fromthe DSP composition on the basis of binding affinity toalpha-1-antitrypsin, apolipoprotein A-I, alpha-1-B-glycoprotein,apolipoprotein A-IV, apolipoprotein D, and prealbumin.

A therapeutic agent may be a small organic molecule or a biologicalmacromolecule, and the specific tissue may be brain, lung, or livertissue. The peptide tag may be coupled to the therapeutic agent by acovalent bond, inclusion complexes, ionic bonds, or hydrogen bonds.Examples of therapeutic agents useful for the practice of this inventionare anti-tumor agents including antimetabolites, cytokine and growthfactor inhibitors, kinase inhibitors, antiangiogenesis agents,anti-inflammatory agents, disease specific antibodies, vaccines, andantibiotics.

Standard immunological, biochemical, and molecular biology methods maybe used herein and are known in the art. Examples of standard protocolscan be found in, for example, Current Protocols series published by JohnWiley and Sons, and all updates available to date, including CurrentProtocols in Molecular Biology, in Immunology, in Cell Biology, inProtein Chemistry, in Pharmacology, and others. All references andpatents and patent applications cited herein are incorporated byreference in their entirety.

EXAMPLES Example 1 Detection of P1-2301 and Cop-1 in Normal Human Serum

PI-2301 (a YFAK random sequence polymer) or Cop-1 (a YEAK randomsequence polymer) were made up at a concentration of 500 ng/mL and werediluted in 5% normal human serum in PBS to concentrations of 100 ng/mL,50 ng/mL, 25 ng/mL, or 12.5 ng/mL, and added to normal human serum.Binding of PI-2301 or Cop-1 to serum proteins contained in the normalhuman serum was detected by addition of rabbit anti-YFAK or rabbitanti-YEAK antibodies.

An uncoated ELISA plate was blocked with PBS/0.1% Tween 20 for 2 hoursat room temperature. PI-2301 or Cop-1 samples were serially diluted inPBS/5% normal human serum and added to the blocked and washed wells ofthe ELISA plate. The PI-2301 or Cop-1 in normal human serum was bound tothe plate and unbound PI-2301 or Cop-1 was removed by washing the platewith PBS/0.05% Tween 20. Protein-A-purified anti-rabbit anti-PI-2301 oranti-rabbit anti-Cop-1, diluted to a suitable concentration based on thetiter, was added for 1 hr at RT. After another wash step to remove theunbound rabbit anti-2301 or rabbit anti-Cop-1 antibodies, a secondaryantibody, a goat anti-rabbit IgG-HRP (horse radish peroxidase conjugatedantibody to rabbit IgG) was added to the well. After washing away anyunbound secondary antibody, substrate for HRP was added to the wells andincubated for 15 minutes, which yielded a blue color that turns yellowwhen stop solution is added, the intensity of which color correlateswith the amount of total PI-2301 or Cop-1 in the well. The opticaldensity was measured at 450 nm with a ELISA plate reader and a titercurve was generated for each set of the serum samples spiked withPI-2301 and Cop-1, respectively. The limit of serum PI-2301 or serumCop-1 detection is defined as the concentration which results in an A450nm absorption which is 3 times above background. ELISA plate wells usedto determine background are treated as described above except PI-2301 orCop-1 was omitted.

Results are plotted in FIG. 2. On the x-axis, the concentration ofcomplex peptide mixture is indicated. On the y-axis, the A450colorimetric absorbance of HRP conjugated secondary antibodies is shown.At higher concentrations of complex peptide mixtures, the detection ofconjugates by anti-PI-2301 or anti-Cop-1 antibodies is higher than lowerconcentrations of complex peptide mixtures. 12.5 ng/mL corresponds to adose of approximately 2 mg in a human patient.

Example 2 Capture of Complexes on a Column

Immobilized PI-2301 or Cop-1 was prepared by reacting the peptides withCNBr-activated Sepharose®, a pre-activated large pore chromatographymedium used for immobilizing ligands (proteins, peptides, nucleic acids)containing primary amines using the cyanogens bromide method. Briefly,after weighing out the desired amount, the freeze-dried CNBr-Sepharose®was washed 10×15 minutes with cold 1 mM HCl (use approximately 200 mL 1mM HCl/gram dried Sepharose) then 2× with coupling buffer. The ligandwas dissolved in coupling buffer to the desired concentration, combinedwith the CNBr-Sepharose® in a 1:2 ratio (use 1 volume of ligand to 2volumes of washed CNBr-Sepharose® gel) then incubated overnight at 4° C.on a rocking platform. Any remaining active sites on the gel wereblocked and then washed to remove any excess ligand. To purify theligand-specific protein, the coupled gel was washed 2× inphosphate-buffered saline (PBS), the desired reagent (serum, cellsupernatant) was added in a 1:2 ratio (1 volume of reagent to 2 volumesof washed CNBr-Sepharose® gel) then incubated overnight at 4° C. on arocking platform. The gel/reagent slurry was packed into a disposablecolumn, washed to remove unbound reagent, then the ligand-specificprotein was eluted with a low pH buffer. After pH neutralization, theabsorbance at 280 nm of the eluted fractions was read to identifyfractions containing the ligands. The column was washed and stored at 4°C. for repeated use.

Example 3 Identification of Proteins Bound to PI-2301 or Cop-1

Samples containing PI-2301 binding proteins or Cop-1 binding proteinswere obtained by the method of Example 1 or Example 2. These sampleswere then enzymatically digested and analyzed by liquid chromatographytandem mass spectrometry (LC-MS/MS) for the purpose of identify theproteins which bind PI-2301 or Cop-1. Briefly, an aliquot of each samplewas digested with the sequence specific protease, trypsin. Afterdigestion, the protein peptide mixture was analyzed by LC-MS/MS.Peptides were separate based on their retention to a release phasecolumn and then sprayed into a mass spectrometer. During the sprayingprocess the peptide picked up a +2 or +3 charge and the massspectrometer monitors the mass overcharge ratio. If a peptide has asignificant mass overcharge ratio it is then fragmented by collisionwith gases and the fragment patterns are recorded. These fragmentpatterns can then be compared to the theoretical fragment patterns ofall known proteins. This molding of experimental fragment patterns totheoretical fragment patterns resulted in the identification of severallipoproteins from the HDL and LDL complexes. These lipoproteins werefound both in the PI-2301 sample and the Cop-1 sample. The Cop-1 samplealso had some unique proteins including complement proteins such as C3and C4A.

FIG. 3 summarizes the serum proteins in normal mouse serum or normalhuman serum which were identified by binding to PI-2301 or Cop-1.PI-2301 may be acetylated or non-acetylated. The sample proteins wereobtained in a method similar to that of Example 1, wherein the PI-2301or Cop-1 were mixed and bound to components in serum. Binding complexesof PI-2301 or Cop-1 were recognized by anti-YFAK or anti-YEAKantibodies, and detected with secondary antibodies and detectionreagents. Serum proteins were eluted from the complex and identified.Proteins are assigned a score based on the A450 absorbance of thedetection reagent. A score of 70 corresponds to a significance value ofp<0.001, as compared to background absorbance, and is consideredstatistically significant.

While capture peptides identified by the method above are expected tobind to DSPs, the above assay may also be performed with DSPs in orderto empirically identify the strongest DSP-binding serum proteins.

Example 4 Comparison of Peptides Composition Across Various Lengths &Lots of DSP Compositions

Following synthesis of different DSP compositions, for example by solidphase synthesis or by solution phase synthesis, the individual lots orbatches made by the same manufacturing process, and individual batchesof mixtures manufactured by different processes may be tested andcompared for variation using bioassays. Depending on the indication ofthe DSP composition, appropriate bioassays include the release of CCL22by the monocyte cell line RAW264.7, ex vivo proliferation assays, andmeasuring the binding of serum proteins to peptides in the DSPcomposition. Using these bioassays, one may determine subsets ofpeptides or even individual peptides that are present in any givenprocess or lot. Processes and lots of DSP compositions will be comparedto determine whether the same subsets of peptides and/or types ofpeptides are consistently represented across the different processes andlots.

A plurality of identifying resins are prepared by immobilizing aselection of serum proteins on solid support. In some embodiments, thecapture protein is a protein of FIG. 3. Each solid support will containat least one serum protein, and if more than one serum protein is boundto the solid support, then the ratio of the individual serum proteinsbound to a given solid support will be consistent across eachidentifying resin. An aliquot from each lot of the DSP composition willbe applied to its own solid support, under conditions that allow asubset of DSPs to bind to the serum proteins. After washing away unboundpeptides, the bound peptides will be eluted. The DSP peptides isolatedin this manner will be further characterized for (1) presence ofdistinct DSP peptides, (2) ratios of peptides to one another, (3)proportion of peptides that bind to the serum binding protein, relativeto the total DSP composition, (4) presence of binding motifs and peptidesequences, (5) amino acid composition and ratios of amino acids, and/orother characteristics of peptides. The characteristics of isolated DSPpeptides from each lot will be compared with each other.

1. A method for detecting a DSP composition, comprising: a. providing asubstantially pure preparation of one or more capture polypeptides; b.affixing the one or more capture polypeptides to a means forquantitatively detecting the DSP composition; and c. determining bindingof the DSP composition to the one or more said capture polypeptide.
 2. Amethod for improving the design of a DSP composition, comprising: a.providing a substantially pure preparation of one or more capturepolypeptides; b. affixing the one or more capture polypeptides to ameans for quantitatively detecting the DSP composition; c. determiningbinding of the DSP composition to the one or more said capturepolypeptides; d. adjusting the design of the DSP composition to eitherenhance or reduce binding to one or more capture polypeptides; e.repeating step (c); f. optionally repeating steps (c-e), whereinadjusting the design of said DSP composition results in any one or moreof: increased bioavailability, reduction in toxicity, and increase inefficacy.
 3. A method for detecting species within a DSP composition,comprising: a. providing a substantially pure preparation of one or morecapture polypeptides; b. affixing the one or more capture polypeptidesto a solid support; c. contacting the solid support with the DSPcomposition; and d. determining binding of individual species of the DSPcomposition to the solid support.
 4. A method for improving the designof species within a DSP composition, comprising: a. providing asubstantially pure preparation of one or more capture polypeptides; b.affixing the one or more capture polypeptides to a solid support; c.contacting the solid support with the DSP composition; d. determiningbinding of individual species of the DSP composition to the solidsupport; e. adjusting the design of the DSP composition to eitherenhance or reduce binding to one or more capture polypeptides; f.repeating step (d); g. optionally repeating steps (d-f), whereinadjusting the design of species of said DSP composition results in anyone or more of: increased bioavailability, reduction in toxicity, andincrease in efficacy.
 5. The method of claim 1, wherein the one or morecapture polypeptides of (a) are identified by: i. affixing the DSPcomposition to a solid support; ii. contacting said solid support in (i)with a protein-containing biological fluid; iii. identifying theproteins from (ii) specifically bound to the solid support in (i).wherein a protein identified in (ii) is a capture polypeptide.
 6. Themethod of claim 1, wherein the capture polypeptide is selected fromcomplement component C3, apolipoprotein A-1 preproprotein,apolipoprotein A-II preproprotein (apolipoprotein D), complementcomponent C4A, trypsin inhibitor, inter-alpha-trypsin inhibitor familyheavy chain-related protein (IHRP), alpha-1-B-glycoprotein,alpha-1-antitrypsin, apolipoprotein A-IV, ceruloplasmin, unnamed proteinproduct (NCBI Locus/Accession No. CAA34971), apolipoprotein E,complement factor B, prealbumin, apolipoprotein C-III, alpha2-HSglycoprotein, apolipoprotein J precursor, Chain C, Immunoglobulin M,immunoglobulin lambda light chain, Coagulation factor II (thrombin), Igkappa chain V-III (KAU cold agglutinin), apolipoprotein J precursor, IgA1 Bur, histidine-rich glycoprotein precursor, Alpha-2-HS-glycoprotein,gelsolin isoform a precursor, inhibitor Kunitz type proteinase, unnamedprotein product (NCBI Locus/Accession No. CAA28659), and Ig J-chain. 7.A method for determining the presence of a DSP composition comprisingthe steps: a. affixing one or more proteins selected from complementcomponent C3, apolipoprotein A-1 preproprotein, apolipoprotein A-IIpreproprotein (apolipoprotein D), complement component C4A, trypsininhibitor, inter-alpha-trypsin inhibitor family heavy chain-relatedprotein (IHRP), alpha-1-B-glycoprotein, alpha-1-antitrypsin,apolipoprotein A-IV, ceruloplasmin, unnamed protein product (NCBILocus/Accession No. CAA34971), apolipoprotein E, complement factor B,prealbumin, apolipoprotein C-III, alpha2-HS glycoprotein, apolipoproteinJ precursor, Chain C, Immunoglobulin M, immunoglobulin lambda lightchain, Coagulation factor II (thrombin), Ig kappa chain V-III (KAU coldagglutinin), apolipoprotein J precursor, Ig A1 Bur, histidine-richglycoprotein precursor, Alpha-2-HS- glycoprotein, gelsolin isoform aprecursor, inhibitor Kunitz type proteinase, unnamed protein product(NCBI Locus/Accession No. CAA28659), and Ig J-chain to a means forquantitatively detecting said DSP composition in a sample; and b.determining the level of said DSP composition in said sample.
 8. Themethod of claim 1 wherein a capture polypeptide is selected fromcomplement component C3, apolipoprotein A-1 preproprotein,apolipoprotein A-II preproprotein (apolipoprotein D), complementcomponent C4A, trypsin inhibitor, inter-alpha-trypsin inhibitor familyheavy chain-related protein (IHRP), alpha-1-B-glycoprotein,alpha-1-antitrypsin, apolipoprotein A-IV, ceruloplasmin, unnamed proteinproduct (NCBI Locus/Accession No. CAA34971), apolipoprotein E,complement factor B, prealbumin, apolipoprotein C-III, alpha2-HSglycoprotein, apolipoprotein J precursor, Chain C, Immunoglobulin M,immunoglobulin lambda light chain, Coagulation factor II (thrombin), Igkappa chain V-III (KAU cold agglutinin), apolipoprotein J precursor, IgA1 Bur, histidine-rich glycoprotein precursor, Alpha-2-HS-glycoprotein,gelsolin isoform a precursor, inhibitor Kunitz type proteinase, unnamedprotein product (NCBI Locus/Accession No. CAA28659), and Ig J-chain. 9.A method for detecting presence of a DSP composition in a biologicalsample, comprising: (a) contacting the biological sample with at leastone capture polypeptide contained in normal human sera, normal non-humanprimate sera, normal rabbit sera, normal mouse sera, normal rat sera,normal ferret sera, normal pig sera, normal dog sera, normal horse sera,normal sheep sera, normal cow sera; and (b) detecting the presence orabsence of binding of the capture polypeptide to the DSP composition,wherein the presence of binding indicates the presence of DSPcomposition in the biological sample.
 10. The method of claim 9, whereinthe capture polypeptide is selected from a polypeptide comprising atleast one component of the HDL proteome, LDL proteome, or at least oneserum protein.
 11. A method for detecting the presence of a DSPcomposition comprising YFAK or YEAK peptides in a biological sample,comprising: (a) contacting the biological sample with at least onecapture polypeptide comprising a peptide selected from: complementcomponent C3, apolipoprotein A-1 preproprotein, apolipoprotein A-IIpreproprotein (apolipoprotein D), complement component C4A, trypsininhibitor, inter-alpha-trypsin inhibitor family heavy chain-relatedprotein (IHRP), alpha-1-B-glycoprotein, alpha-1-antitrypsin,apolipoprotein A-IV, ceruloplasmin, unnamed protein product (NCBILocus/Accession No. CAA34971), apolipoprotein E, complement factor B,prealbumin, apolipoprotein C-III, alpha2-HS glycoprotein, apolipoproteinJ precursor, Chain C, Immunoglobulin M, immunoglobulin lambda lightchain, Coagulation factor II (thrombin), Ig kappa chain V-III (KAU coldagglutinin), apolipoprotein J precursor, Ig A1 Bur, histidine-richglycoprotein precursor, Alpha-2-HS- glycoprotein, gelsolin isoform aprecursor, inhibitor Kunitz type proteinase, unnamed protein product(NCBI Locus/Accession No. CAA28659), and Ig J-chain; and (b) detectingthe presence or absence of binding of the capture polypeptide to the DSPcomposition, wherein the presence of binding indicates the presence ofYFAK or YEAK peptides in the biological sample.
 12. A method formeasuring an amount of a DSP composition comprising YFAK or YEAKpeptides in a biological sample, comprising: (a) contacting thebiological sample with at least one capture polypeptide comprising apeptide selected from complement component C3, apolipoprotein A-1preproprotein, apolipoprotein A-II preproprotein (apolipoprotein D),complement component C4A, trypsin inhibitor, inter-alpha-trypsininhibitor family heavy chain-related protein (IHRP),alpha-1-B-glycoprotein, alpha-1-antitrypsin, apolipoprotein A-IV,ceruloplasmin, unnamed protein product (NCBI Locus/Accession No.CAA34971), apolipoprotein E, complement factor B, prealbumin,apolipoprotein C-III, alpha2-HS glycoprotein, apolipoprotein Jprecursor, Chain C, Immunoglobulin M, immunoglobulin lambda light chain,Coagulation factor II (thrombin), Ig kappa chain V-III (KAU coldagglutinin), apolipoprotein J precursor, Ig A1 Bur, histidine-richglycoprotein precursor, Alpha-2-HS- glycoprotein, gelsolin isoform aprecursor, inhibitor Kunitz type proteinase, unnamed protein product(NCBI Locus/Accession No. CAA28659), and Ig J-chain; (b) quantifying alevel of binding of the capture polypeptide to the DSP composition;wherein the level of binding indicates the amount of the DSP compositionin the biological sample.
 13. A method for measuring bioavailability ofa DSP composition in a mammal, comprising: (a) administering to a mammala dose of a composition comprising the DSP composition; (b) removing abiological sample from the subject; and (c) contacting the biologicalsample with at least one capture polypeptide comprising a peptideselected from complement component C3, apolipoprotein A-1 preproprotein,apolipoprotein A-II preproprotein (apolipoprotein D), complementcomponent C4A, trypsin inhibitor, inter-alpha-trypsin inhibitor familyheavy chain-related protein (IHRP), alpha-1-B-glycoprotein,alpha-1-antitrypsin, apolipoprotein A-IV, ceruloplasmin, unnamed proteinproduct (NCBI Locus/Accession No. CAA34971), apolipoprotein E,complement factor B, prealbumin, apolipoprotein C-III, alpha2-HSglycoprotein, apolipoprotein J precursor, Chain C, Immunoglobulin M,immunoglobulin lambda light chain, Coagulation factor II (thrombin), Igkappa chain V-III (KAU cold agglutinin), apolipoprotein J precursor, IgA1 Bur, histidine-rich glycoprotein precursor, Alpha-2-HS- glycoprotein,gelsolin isoform a precursor, inhibitor Kunitz type proteinase, unnamedprotein product (NCBI Locus/Accession No. CAA28659), and Ig J-chain;thereby determining the bioavailability of the DSP composition in thebiological sample.
 14. A method for determining a suitable dose of a DSPcomposition to administer to a subject in need thereof, comprising: (a)administering to the subject a dose of the DSP composition; (b) removinga biological sample from the subject; (c) contacting the biologicalsample with at least one capture polypeptide comprising a peptideselected from complement component C3, apolipoprotein A-1 preproprotein,apolipoprotein A-II preproprotein (apolipoprotein D), complementcomponent C4A, trypsin inhibitor, inter-alpha-trypsin inhibitor familyheavy chain-related protein (IHRP), alpha-1-B-glycoprotein,alpha-1-antitrypsin, apolipoprotein A-IV, ceruloplasmin, unnamed proteinproduct (NCBI Locus/Accession No. CAA34971), apolipoprotein E,complement factor B, prealbumin, apolipoprotein C-III, alpha2-HSglycoprotein, apolipoprotein J precursor, Chain C, Immunoglobulin M,immunoglobulin lambda light chain, Coagulation factor II (thrombin), Igkappa chain V-III (KAU cold agglutinin), apolipoprotein J precursor, IgA1 Bur, histidine-rich glycoprotein precursor, Alpha-2-HS- glycoprotein,gelsolin isoform a precursor, inhibitor Kunitz type proteinase, unnamedprotein product (NCBI Locus/Accession No. CAA28659), and Ig J-chain; (d)determining a level of the capture polypeptide in the biological sample;(e) optionally repeating steps (a) through (d) using a different dose;and (f) comparing the levels to a predetermined suitable level of theDSP composition in the biological sample; wherein the suitable dose isthe dose that results in the predetermined suitable level of the DSPcomposition in the biological sample.
 15. A method for treating orpreventing an unwanted immune response in a subject, comprising: (a)administering to the subject a suitable dose of a DSP composition,wherein such suitable dose is determined by: (i) administering to thesubject a dose of the DSP composition; (ii) removing a biological samplefrom the experimental subject; (iii) contacting the biological samplewith at least one capture polypeptide selected from complement componentC3, apolipoprotein A-1 preproprotein, apolipoprotein A-II preproprotein(apolipoprotein D), complement component C4A, trypsin inhibitor,inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP),alpha-1-B-glycoprotein, alpha-1-antitrypsin, apolipoprotein A-IV,ceruloplasmin, unnamed protein product (NCBI Locus/Accession No.CAA34971), apolipoprotein E, complement factor B, prealbumin,apolipoprotein C-III, alpha2-HS glycoprotein, apolipoprotein Jprecursor, Chain C, Immunoglobulin M, immunoglobulin lambda light chain,Coagulation factor II (thrombin), Ig kappa chain V-III (KAU coldagglutinin), apolipoprotein J precursor, Ig A1 Bur, histidine-richglycoprotein precursor, Alpha-2-HS-glycoprotein, gelsolin isoform aprecursor, inhibitor Kunitz type proteinase, unnamed protein product(NCBI Locus/Accession No. CAA28659), and Ig J-chain; (iv) determining alevel of the capture polypeptide in the biological sample; (v)optionally repeating steps (i) through (iv) using a different dose; and(vi) comparing the level(s) against a predetermined suitable level ofthe DSP composition in the biological sample; wherein a suitable dose isthe dose that results in the predetermined suitable level of the DSPcomposition in said biological sample.
 16. The method of claim 11,wherein the capture polypeptide is labeled.
 17. The method of claim 11,wherein the capture polypeptide is affixed to a solid support.
 18. Themethod of claim 11, further comprising isolating a complex comprisingthe capture polypeptide bound to the DSP composition.
 19. The method ofclaim 11, further comprising detecting binding of the capturepolypeptide to the DSP composition with antibodies to the capturepolypeptide.
 20. The method of claim 11, wherein the composition isadministered subcutaneously.
 21. A composition for detecting a DSPcomposition in a biological sample, comprising at least one capturepolypeptide comprising a peptide selected from complement component C3,apolipoprotem A-1 preproprotein, apolipoprotem A-II preproprotein(apolipoprotem D), complement component C4A, trypsin inhibitor,inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP),alpha-1-B-glycoprotein, alpha-1-antitrypsin, apolipoprotem A-IV,ceruloplasmin, unnamed protein product (NCBI Locus/Accession No.CAA34971), apolipoprotem E, complement factor B, prealbumin,apolipoprotem C-III, alpha2-HS glycoprotein, apolipoprotein J precursor,Chain C, Immunoglobulin M, immunoglobulin lambda light chain,Coagulation factor II (thrombin), Ig kappa chain V-III (KAU coldagglutinin), apolipoprotein J precursor, Ig A1 Bur, histidine-richglycoprotein precursor, Alpha-2-HS-glycoprotein, gelsolin isoform aprecursor, inhibitor Kunitz type proteinase, unnamed protein product(NCBI Locus/Accession No. CAA28659), and Ig J-chain.
 22. A method forisolating peptides from a sample comprising a DSP composition,comprising: (a) contacting the sample with at least one capturepolypeptide comprising a peptide selected from complement component C3,apolipoprotein A-1 preproprotein, apolipoprotein A-II preproprotein(apolipoprotein D), complement component C4A, trypsin inhibitor,inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP),alpha-1-B-glycoprotein, alpha-1-antitrypsin, apolipoprotein A-IV,ceruloplasmin, unnamed protein product (NCBI Locus/Accession No.CAA34971), apolipoprotein E, complement factor B, prealbumin,apolipoprotein C-III, alpha2-HS glycoprotein, apolipoprotein Jprecursor, Chain C, Immunoglobulin M, immunoglobulin lambda light chain,Coagulation factor II (thrombin), Ig kappa chain V-III (KAU coldagglutinin), apolipoprotein J precursor, Ig A1 Bur, histidine-richglycoprotein precursor, Alpha-2-HS-glycoprotein, gelsolin isoform aprecursor, inhibitor Kunitz type proteinase, unnamed protein product(NCBI Locus/Accession No. CAA28659), and Ig J-chain; and (b) separatingpeptides that bind to the capture polypeptide from the mixture.
 23. Themethod of claim 22, wherein the capture polypeptide is immobilized onsolid support.
 24. The method of claim 23, wherein the capturepolypeptide is epitope-tagged.
 25. The method of claim 22, furthercomprising separating bound peptides from the capture polypeptides. 26.The method of claim 22, further comprising determining characteristicsof isolated peptides.
 27. The method of claim 26, wherein determiningcharacteristics comprises determining an amino acid sequence of a boundpeptide or determining relative ratios of amino acids in bound peptides.28. A method for identifying bioavailable peptides in a DSP compositionin a subject, comprising: (a) administering the DSP composition to thesubject at a first time; and (b) at a second time after administration,removing a tissue sample from the patient; and (c) identifying peptidesin the sample that bind to at least one capture polypeptide comprising apeptide selected from complement component C3, apolipoprotein A-1preproprotein, apolipoprotein A-II preproprotein (apolipoprotein D),complement component C4A, trypsin inhibitor, inter-alpha-trypsininhibitor family heavy chain-related protein (IHRP),alpha-1-B-glycoprotein, alpha-1-antitrypsin, apolipoprotein A-IV,ceruloplasmin, unnamed protein product (NCBI Locus/Accession No.CAA34971), apolipoprotein E, complement factor B, prealbumin,apolipoprotein C-III, alpha2-HS glycoprotein, apolipoprotein Jprecursor, Chain C, Immunoglobulin M, immunoglobulin lambda light chain,Coagulation factor II (thrombin), Ig kappa chain V-III (KAU coldagglutinin), apolipoprotein J precursor, Ig A1 Bur, histidine-richglycoprotein precursor, Alpha-2-HS- glycoprotein, gelsolin isoform aprecursor, inhibitor Kunitz type proteinase, unnamed protein product(NCBI Locus/Accession No. CAA28659), and Ig J-chain.
 29. A method forproducing a DSP composition having reduced toxicity, comprising: (a)contacting the DSP composition with at least one capture polypeptidecomprising a peptide selected from complement component C3,apolipoprotein A-1 preproprotein, apolipoprotein A-II preproprotein(apolipoprotein D), complement component C4A, trypsin inhibitor,inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP),alpha-1-B-glycoprotein, alpha-1-antitrypsin, apolipoprotein A-IV,ceruloplasmin, unnamed protein product (NCBI Locus/Accession No.CAA34971), apolipoprotein E, complement factor B, prealbumin,apolipoprotein C-III, alpha2-HS glycoprotein, apolipoprotein Jprecursor, Chain C, Immunoglobulin M, immunoglobulin lambda light chain,Coagulation factor II (thrombin), Ig kappa chain V-III (KAU coldagglutinin), apolipoprotein J precursor, Ig A1 Bur, histidine-richglycoprotein precursor, Alpha-2-HS- glycoprotein, gelsolin isoform aprecursor, inhibitor Kunitz type proteinase, unnamed protein product(NCBI Locus/Accession No. CAA28659), and Ig J-chain; and (b) separatingpeptides that bind to the capture polypeptide from the mixture; (c)determining characteristics of the separated peptides; and (d) preparinga set of peptides with the characteristics of the separated peptides.30. A method for producing a DSP composition having enhanced potency,comprising: (a) contacting the DSP composition with at least one capturepolypeptide comprising a peptide selected from complement component C3,apolipoprotein A-1 preproprotein, apolipoprotein A-II preproprotein(apolipoprotein D), complement component C4A, trypsin inhibitor,inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP),alpha-1-B-glycoprotein, alpha-1-antitrypsin, apolipoprotein A-IV,ceruloplasmin, unnamed protein product (NCBI Locus/Accession No.CAA34971), apolipoprotein E, complement factor B, prealbumin,apolipoprotein C-III, alpha2-HS glycoprotein, apolipoprotein Jprecursor, Chain C, Immunoglobulin M, immunoglobulin lambda light chain,Coagulation factor II (thrombin), Ig kappa chain V-III (KAU coldagglutinin), apolipoprotein J precursor, Ig A1 Bur, histidine-richglycoprotein precursor, Alpha-2-HS-glycoprotein, gelsolin isoform aprecursor, inhibitor Kunitz type proteinase, unnamed protein product(NCBI Locus/Accession No. CAA28659), and Ig J-chain; and (b) separatingpeptides that bind to the capture polypeptide from the mixture; (c)determining characteristics of the separated peptides; and preparing aset of peptides with the characteristics of the separated peptides. 31.A method for treating or preventing an unwanted immune response in asubject, comprising: (a) providing a DSP composition; (b) administeringthe DSP composition to a test subject; (c) removing a biological samplefrom the test subject; (d) contacting the biological sample with atleast one capture polypeptide comprising a peptide sequence selectedfrom complement component C3, apolipoprotein A-1 preproprotein,apolipoprotein A-II preproprotein (apolipoprotein D), complementcomponent C4A, trypsin inhibitor, inter-alpha-trypsin inhibitor familyheavy chain-related protein (IHRP), alpha-1-B-glycoprotein,alpha-1-antitrypsin, apolipoprotein A-IV, ceruloplasmin, unnamed proteinproduct (NCBI Locus/Accession No. CAA34971), apolipoprotein E,complement factor B, prealbumin, apolipoprotein C-III, alpha2-HSglycoprotein, apolipoprotein J precursor, Chain C, Immunoglobulin M,immunoglobulin lambda light chain, Coagulation factor II (thrombin), Igkappa chain V-III (KAU cold agglutinin), apolipoprotein J precursor, IgA1 Bur, histidine-rich glycoprotein precursor, Alpha-2-HS-glycoprotein,gelsolin isoform a precursor, inhibitor Kunitz type proteinase, unnamedprotein product (NCBI Locus/Accession No. CAA28659), and Ig J-chain; (e)separating peptides that bind to the capture polypeptide from themixture; (f) determining characteristics of the separated peptides; (g)preparing a set of peptides with the characteristics of the separatedpeptides, and (h) administering the new set of peptides to a subject.32. The method of claim 28, wherein the peptides are administered to thesubject more than once.
 33. The method of claim 32, wherein the peptidesare administered to the subject at intervals of 1, 2, 3, 4, 6, 12, 18,24, 36, 48, or 72 hours.
 34. A method for comparing differentpreparations of DSP composition, comprising: (a) contacting a first DSPcomposition with at least one capture polypeptide contained in normalhuman sera, normal non-human primate sera, normal rabbit sera, normalmouse sera, normal rat sera, normal ferret sera, normal pig sera, normaldog sera, normal horse sera, normal sheep sera, normal cow sera; and (b)contacting a second DSP composition with at least one capturepolypeptide comprising a peptide selected from: normal human sera,normal non-human primate sera, normal rabbit sera, normal mouse sera,normal rat sera, normal ferret sera, normal pig sera, normal dog sera,normal horse sera, normal sheep sera, normal cow sera; and (c) repeatingstep (b) as necessary; and (d) separating peptides that bind to thecapture polypeptide from the mixtures from steps (a-c); (e) determiningcharacteristics of the separated peptides from step (d); and (f)comparing said separated set of peptides with the characteristics of theseparated peptides from step (d).
 35. A method for preparing atherapeutic agent to a target tissue in a subject, comprising: (a)providing a DSP composition; and (b) coupling a therapeutic agent to theDSP composition to form a conjugate.
 36. A method for delivering atherapeutic agent to a specific tissue in a subject, comprising: (a)isolating a peptide tag by: (i) contacting a DSP composition with atissue-specific peptide comprising a peptide selected from complementcomponent C3, apolipoprotein A-1 preproprotein, apolipoprotein A-IIpreproprotein (apolipoprotein D), complement component C4A, trypsininhibitor, inter-alpha-trypsin inhibitor family heavy chain-relatedprotein (IHRP), alpha-1-B-glycoprotein, alpha-1-antitrypsin,apolipoprotein A-IV, ceruloplasmin, unnamed protein product (NCBILocus/Accession No. CAA34971), apo lipoprotein E, complement factor B,prealbumin, apolipoprotein C-III, alpha2-HS glycoprotein, apolipoproteinJ precursor, Chain C, Immunoglobulin M, immunoglobulin lambda lightchain, Coagulation factor II (thrombin), Ig kappa chain V-III (KAU coldagglutinin), apolipoprotein J precursor, Ig A1 Bur, histidine-richglycoprotein precursor, Alpha-2-HS-glycoprotein, gelsolin isoform aprecursor, inhibitor Kunitz type proteinase, unnamed protein product(NCBI Locus/Accession No. CAA28659), and Ig J-chain; and (ii) separatingpeptides that bind to the tissue-specific peptide from the mixture; (b)coupling the peptide tag to a therapeutic agent; and (c) administeringthe conjugate to a subject.
 37. The method of claim 35, wherein thetherapeutic agent is a small organic molecule or a biologicalmacromolecule.
 38. The method of claim 35, wherein the tissue is brain,lung, or liver tissue.
 39. The method of claim 35, wherein the peptidetag is coupled to the therapeutic agent by a covalent bond, inclusioncomplexes, ionic bonds, or hydrogen bonds.